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DNA Methylation Machinery Mediates the Bladder’s Response to Obstruction
by
Jia Xin Jiang
A thesis submitted in conformity with the requirements for the degree of Masters of Science
Department of Physiology University of Toronto
© Copyright by Jia-Xin Jiang 2015
ii
DNA Methylation Machinery Mediates Bladder’s Response to
Obstruction
Jia-Xin Jiang
Master of Science
Department of Physiology
University of Toronto
2015
Abstract
Partial bladder obstruction arises insidiously and compromises quality of life often by
perpetuating urinary tract infections, incontinence and kidney failure. Obstruction leads to
prolonged over-distension of the bladder, which induces tissue hypoxia and abnormal
extracellular matrix (ECM) remodeling that alters bladder smooth muscle cell (BSMC)
phenotype. The BSMCs become hypertrophic and proliferative with loss of differentiation and
contractility. The persistence of pathology despite de-obstruction in human and animal bladders
in vivo, as well as the stable pheno-pathology found in BSMC in vitro, suggests that responses to
bladder obstruction are mediated by the microenvironment. We hypothesize that epigenetic
mechanisms mediate irreversible BSMC responses to the obstructive environment. In animals,
long-term bladder obstruction significantly increased bladder weight and dysregulated gene
expressions, neither were completely reversed by de-obstruction. The ECM regulates BSMC
phenotype switching in vitro, which is mediated by alterations in the expression as well as the
sub-cellular localization of DNA Methyl-Transferase.
iii
To my grandparents, Xue Qing Jiang and Hong Zhu, thank you for building the foundation of my happiness.
iv
Acknowledgments
First, I want to express the deepest gratitude to my mother, I am constantly nurtured and
strengthened by her unconditional love. I would like to thank my supervisor Dr. Darius Bagli, a
brilliant scientist and an excellent mentor, for his guidance, encouragement and patience. I am
greatly indebted to Dr. Karen Aitken, a teacher as well as a friend to me, for her expertise and
caring support during the course of my studies. I am grateful to my supervisory committee
members, Drs. Scott Heximer, Robert Jankov and Rossana Weksberg, who have provided me
with valuable advices and a great deal of support when I faced challenges. A special thank you to
Tyler Kirwan and Youan Liu, who helped me with many technical aspects of the experiments, it
has been such a privilege and pleasure to work with them. I am thankful to my dear friend, Hao
Wang, for listening to me and being there for me always.
I want to thank the department of Physiology, the University of Toronto Fellowship award, the
RESTRACOMP (research training award at the Hospital for Sick Children) and the Ontario
Graduate Scholarship for their training and academic support.
Contributions
Many people have contributed to this thesis: Trupti conducted the cell proliferation experiment
on different collagen matrices; As mentioned, Tyler and Youan had helped with many technical
aspects of my experiments; Dr. Schroder performed the animal surgery in the first in vivo study
(study 1, Chapter 3); Youliang Liu performed the pyrosequencing experiments (Chapter 4);
Lastly, Stephanie Beadman helped with the smooth muscle cell transfection experiment
(Appendix I).
v
Table of Contents
Acknowledgments.......................................................................................................................... iii
Table of Contents .............................................................................................................................v
List of Tables ................................................................................................................................. ix
List of Figures ..................................................................................................................................x
1 Chapter 1 Introduction to the bladder and epigenetics................................................................1
1.1 The urinary bladder and its function ....................................................................................1
1.2 Bladder obstruction ..............................................................................................................2
1.2.1 Obstruction leads to bladder hypertrophy ................................................................2
1.2.2 Matrix deposition causes the loss of compliance and leads to bladder
overactivity ..............................................................................................................3
1.2.3 Incomplete anatomical and functional reversal even after intervention ..................4
1.2.4 Current treatments ....................................................................................................5
1.2.5 Three inciting stimuli during obstruction progression: stretch, hypoxia and
damaged matrix ........................................................................................................8
1.3 Smooth muscle cells ............................................................................................................9
1.3.1 Smooth muscle plasticity .......................................................................................10
1.3.2 SMC phenotypic modulation and diseases ............................................................11
1.4 The extracellular matrix .....................................................................................................12
1.4.1 Matrix and cell phenotype regulation ....................................................................12
1.4.2 The extracellular matrix of the normal and obstructed bladder .............................13
1.4.3 Matrix regulation of smooth muscle phenotype ....................................................14
1.5 Epigenetics .........................................................................................................................15
1.5.1 Mechanisms of epigenetic regulation ....................................................................15
1.5.2 Epigenetic modulation of SMC phenotype ............................................................17
1.6 Hypothesis and Aims .........................................................................................................18
vi
1.6.1 Hypothesis..............................................................................................................18
1.6.2 Specific aims and experimental plans ....................................................................18
2 Chapter 2: Smooth Muscle Cell Phenotypic Switching Induced by Damaged Matrix Is
Associated with Changes in DNA Methylation ........................................................................20
2.1 Methods..............................................................................................................................20
2.1.1 Bladder Smooth Muscle Cell (BSMC) culture ......................................................20
2.1.2 Primary huBSMC transfection ...............................................................................20
2.1.3 Preparation of Collagen Substrates ........................................................................21
2.1.4 Hypoxia ..................................................................................................................21
2.1.5 Immunostaining and Confocal Microscopy ...........................................................21
2.1.6 Cell Counting .........................................................................................................22
2.1.7 Protein Extraction methods and Western blotting .................................................22
2.1.8 RNA extraction, Reverse Transcription and Polymerase Chain Reaction (PCR)
................................................................................................................................23
2.1.9 Agarose gel electrophoresis ...................................................................................23
2.1.10 Drugs and treatments .............................................................................................24
2.1.11 Illumina Bead-chip analysis of DNA methylation on damaged matrix .................24
2.1.12 Statistics .................................................................................................................25
2.2 Results ................................................................................................................................26
2.2.1 Damaged Matrix-induced cell proliferation and de-differentiation is dependent
on DNMT activity ..................................................................................................26
2.2.2 Matrix alters intracellular DNA methyltransferase 3A (DNMT3A) localization
and expression in visceral smooth muscle cells .....................................................28
2.2.3 Hypoxia potentiates nuclear DNMT3A expression on DNC ................................28
2.2.4 Matrix regulation of DNMT3A depends upon culture duration, transcription
and translation ........................................................................................................31
2.2.5 Increased DNMT3A expression does not alter its localization .............................31
2.2.6 Matrix regulation of DNMT3A is cell density dependent ...............................33
vii
2.2.7 Signaling pathways regulate Dnmt3a localization on damaged matrix .................35
2.2.8 Inhibition of nuclear export did not alter DNMT3A localization on NC and
DNC .......................................................................................................................35
2.2.9 Matrix induces significant changes in DNA methylation ......................................38
3 Chapter 3: Gene expression is persistently altered in irreversible bladder obstruction ............42
3.1 Methods..............................................................................................................................42
3.1.1 Bladder obstruction and release .............................................................................42
3.1.2 RNA and DNA isolation ........................................................................................43
3.1.3 Custom PCR Array (with additional samples).......................................................44
3.1.4 Validation of expression changes ..........................................................................44
3.1.5 Pyrosequencing ......................................................................................................46
3.2 Results ................................................................................................................................47
3.2.1 Release of 6 week bladder obstruction does not completely reverse the
bladder/body weight ratio or functional parameters ..............................................47
3.2.2 6 week bladder obstruction leads to dysregulation of genes that are persistent
even after release....................................................................................................48
3.2.3 Pyrosequencing ......................................................................................................51
4 Chapter 4: Discussion ...............................................................................................................54
4.1 Matrix and SMC biology ...................................................................................................54
4.1.1 Control of DNMT3A localization ..........................................................................54
4.1.2 MMP remodeling during fibroproliferative disease ..............................................55
4.1.3 Epigenetic mediation of disease progression .........................................................57
4.1.4 Matrix alters DNA methylation in bSMC ..............................................................57
4.1.5 Future directions ....................................................................................................58
4.2 Irreversible bladder obstruction .........................................................................................59
4.2.1 Irreversible bladder obstruction and persistent gene dysregulation .......................59
4.2.2 Variability in animal models of bladder obstruction .............................................61
viii
4.2.3 Future directions ....................................................................................................62
5 Table 8: Abbreviations ..............................................................................................................63
References ......................................................................................................................................64
Appendices .....................................................................................................................................78
Appendix I: Human BSMC Transfection ......................................................................................78
Rationale ...................................................................................................................................78
Material and Methods ...............................................................................................................78
HuBSMC transfection ........................................................................................................78
Immunoprecipitation ..........................................................................................................79
Results .......................................................................................................................................79
HuBSMC transfection using Lipofectamine was not efficient ..........................................79
Transfection using the Nucleofector II system lead to higher transfection efficiencies ....81
Serum starvation is required prior to plating onto matrices ...............................................85
Myc-DNMT3A exhibit consistent localization patter with endogenous DNMT3A .........86
Immunoprecipitation and Mass Spectrometry (MS) requires large amount of starting
materials .................................................................................................................87
Discussion and future directions ...............................................................................................89
Optimal transfection conditions .........................................................................................90
Large amount of starting material is required for MS .......................................................90
Future directions ................................................................................................................91
Appendix II: Supplemental Figure and Table ...........................................................................92
ix
List of Tables
Table 1 List of immunofluorescence antibodies ........................................................................... 22
Table 2 List of western antibodies ................................................................................................ 23
Table 3 List of drug treatments ..................................................................................................... 24
Table 4 Differentially methylated CpG sites ................................................................................ 40
Table 5 Sample groups.................................................................................................................. 43
Table 6 Genes with CpG islands, curated from rat bladder obstruction literature ....................... 45
Table 7. Percent methylation at each CpG site ............................................................................ 53
Table 8: Abbreviations .................................................................................................................. 63
Table 9: Gene expression changes from microarray analysis 124 .................................................. 92
x
List of Figures
Figure 1 Duration of obstruction determines the degree of phenotype reversal following de-
obstruction....................................................................................................................................... 8
Figure 2: Proliferation and de-differentiation of SMC on denatured matrix depends on DNMT
activity in visceral smooth muscle cells........................................................................................ 27
Figure 3: Matrix is a critical determinant of DNMT3A expression in visceral smooth muscle
cells. .............................................................................................................................................. 29
Figure 4: Hypoxia and damaged matrix increase DNMT3A nuclear expression in a cooperative
fashion. .......................................................................................................................................... 30
Figure 5: Nuclear Localization of DNMT3A is dependent upon the time after plating and
transcription. ................................................................................................................................. 32
Figure 6 DNMT3A transfection does not alter subcellular localization ....................................... 33
Figure 7: DNMT3A expression is regulated by cell-density, mitosis but not mitogenic growth
factors. ........................................................................................................................................... 34
Figure 8: DNMT3A expression is inhibited by ERK and F11 inhibitors on DNC....................... 36
Figure 9 : Nuclear export inhibitor did not alter DNMT3A localization on NC .......................... 37
Figure 10: Damaged matrix induces DNMT3A nuclear expression in human bladder SMC and
changes in methylation status in CpG sites of the Illumina 450K methylation array. .................. 39
Figure 11: A priori test of CpG sites in SMC specific genes reveals specific changes in DNA
methylation. .................................................................................................................................. 41
Figure 12 Bladder obstruction significantly upregulates bladder mass ........................................ 48
Figure 13: Long term obstruction causes persistently dysregulated gene expression that is not
reversed by de-obstruction ............................................................................................................ 50
xi
Figure 14: DNA methylation states of 7 CpG sites upstream of KCNB2 were not changed ....... 52
Figure 15 Transfection using Lipofectamine ................................................................................ 80
Figure 16 GFP transfection using Lipofectamine ......................................................................... 81
Figure 17 GFP transfection with Nucleofector ............................................................................. 82
Figure 18: Sub-passage decreases plasmid expression ................................................................. 83
Figure 19 : Myc-DNMT3A plasmid expression is highest at 48 hours. ....................................... 85
Figure 20: Plating without prior starvation induces nuclear DNMT3A expression ..................... 86
Figure 21: Timecourse experiment using transfected human BSMC ........................................... 89
Figure 22 : Myc-DNMT3A is detected in the IP product of transfected human BSMCs ............ 89
Figure 23: AKT signaling is upregulated on DNC at 3 hours. ..................................................... 93
Figure 24. CpG island upstream of the human KCNB2 gene....................................................... 94
1
Chapter 1 Introduction to the bladder and epigenetics
1.1 The urinary bladder and its function
The urinary bladder is a unique organ that collects, stores and releases urine. It is remarkably
distensible to accommodate the large increase in its luminal volume during the urine filling phase
and is able to contract in a coordinated manner to expel its contents upon demand. The
mechanical and physical functions of the bladder are modulated by the epithelium, the contractile
smooth muscle and the organ’s extracellular matrix (ECM) component, allowing the bladder to
repeatedly expand and contract to its original size. Derangements with the optimal function of
the bladder smooth muscle and the ECM properties can compromise effective bladder
contraction and urine storage.
The bladder can be divided into two parts: the superior half is referred to as the dome while the
inferior half is referred as the base of the bladder1,2. The dome of the bladder is thinner and more
distensible to accommodate a large increase in volume during urine filling, whereas the base of
the bladder is less distensible 3. Urine enters the bladder from the left and right kidneys via the
openings of the two ureters and exits at the bladder neck through the urethra. The area between
the inlets of ureters and the urethral opening is the trigone1.
The bladder has several distinct tissue layers. The urothelium, together with the lamina propria
makes up the mucosa. The urothelium lines the lumen of the bladder and acts as a barrier
between urine and the underlying tissues1. The outermost layer of the urothelium is made of
large transitional epithelial cells that can flatten for more surface area, in order to accommodate
luminal expansion during bladder filling phase2. The submucosa layer of the bladder consists of
smooth muscle, blood vessel, nerve and connective tissue and delivers the tissue with nutrients
and oxygen supply1. Lastly, the visceral muscle (referred to as the detrusor) is made of bundles
of bladder smooth muscle cells (BSMCs) that are organized into 3 layers3: the inner longitudinal
layer, the middle circular layer and the outer longitudinal layer.
The alternating cycles of urine storage and expulsion requires control by a set of intricate neural
circuits in the cortex, brainstem and the spinal cord as well as the mechanical and physical
2
properties of the contractile smooth muscle cells (SMCs) and the extracellular matrix (ECM).
During the filling phase, the pressure inside the bladder rises minimally with increased luminal
volume3. The smooth muscle must remain elongated for a prolonged time (hours) to maintain the
low pressure before sufficient amount of urine has accumulated. When the threshold volume is
reached, the mechanoreceptors in the urothelium and the detrusor causes increased
parasympathetic input and decreased sympathetic input through the micturition reflexes. This
leads to the relaxation of the urethral sphincters, followed by a coordinated contraction across the
detrusor muscle to allow rapid urine expulsion and therefore bladder emptying4. The
mechanoreceptors also sends signals to the brainstem via the pelvic nerve afferents and the spinal
cord.
1.2 Bladder obstruction
Bladder outlet obstruction is defined as any ailment that disrupts normal urine outflow and can
have anatomical or neurological etiologies. Congenital defects such as the Posterior Urethral
Valve (PUV) in children or Benign Prostate Hyperplasia (BPH) in older males can lead to
physical resistance in the bladder outlet that impedes urine expulsion. Neurogenic bladder
obstruction, on the other hand, is often caused by conditions/ damage to the brain stem or the
spinal cord, resulting in inappropriate neural activation of the bladder. In this case, normal urine
outflow is disrupted either by the failure to properly relax the urethral sphincter or uncoordinated
muscle contraction during maturation.
1.2.1 Obstruction leads to bladder hypertrophy
Regardless of the etiology, bladder obstruction leads to incomplete voiding and urine retention in
the bladder, and thus excessive pressure in the bladder wall and the initiation of subsequent
physiological changes. As obstruction progresses, the bladder undergoes profound changes in
morphology as well as function. Many studies have documented the pathophysiology of bladder
obstruction using animal models5-9.
3
The onset of bladder obstruction is followed by an inflammatory phase and an early
compensatory phase, which is marked by increased muscle mass to cope with the increased
workload due to urine retention and outlet resistance. As obstruction progresses, however, the
bladder becomes de-compensated with decreased contractility and compliance. Metcalfe et al.
has shown that increased expression of important inflammatory mediators such as the CTGF and
TGF-2 weeks post obstruction in rats7. In other animal studies with longer duration of
obstruction, the bladder becomes profoundly hypertrophied, increasing 5 to 10 fold in mass in
rats5,8 and up to 3 fold in mice 6 by the end of 7 and 4 weeks, respectively. The hypertrophy of
the detrusor muscle observed in animal obstruction studies is consistent with the hypertrophy
observed in human bladder obstruction. Moreover, the bladder wall is significantly thickened due
to increased ECM deposition compared to the sham operated animals. Subsequently, the
compensatory phase is followed by the decompensatory phase, during which the bladder suffers
further remodeling and functional decline. In a long-term mouse bladder obstruction study,
severely hypertrophied bladder generated significantly less contractile force despite the increased
muscle mass6. In addition to the failure to generate force, smooth muscle from obstructed bladder
also fails to properly respond to neurotransmitters. In another rodent bladder obstruction study,
muscle strips from severely obstructed bladder (6 weeks post obstruction) failed to relax
following norepinephrine stimulation8.
Similar patterns of pathology progression can also be observed in vascular smooth muscle and
heart10-13. Vascular smooth muscle and heart muscle initially undergoes hypertrophy in response
to increased workload. However, this response was followed by a secondary decompensatory
phase, exhibiting muscle function decline and cell apoptosis despite the persistent hypertrophy14.
Furthermore, altered ECM deposition and remodeling also impacts bladder function.
1.2.2 Matrix deposition causes the loss of compliance and leads to bladder
overactivity
Increased collagen deposition in the bladder is considered as a part of the initial compensatory
response to bladder obstruction. Subsequently, however, this process impacts the physical and
mechanical properties of the bladder. In one study of ECM alterations during bladder obstruction,
the smooth muscle fraction decreased from 69% (sham group) to 61% in hypertrophied bladders
4
due to changes in ECM composition, despite the increase in bladder mass6. Furthermore,
increased matrix deposition profoundly decreases the compliance of the bladder muscle wall.
The quantitative measure of urological compliance is the change in filling volume per change in
pressure. During the filling phase, the maintenance of low pressure is dependent on the
appropriate level of compliance as the muscular wall is stretched. Many patients with bladder
obstruction develop low compliance in the bladder wall15. In a rabbit model of bladder
obstruction, the total amount of collagen was significantly upregulated by obstruction and
lowered muscle wall compliance was also observed16. The mechanical properties of the bladder
wall is sensitive to its own structure and composition2. When the detrusor becomes stiff due to
matrix deposition, a given bladder volume then results in higher intraluminal pressure1. With
increased urine retention (due to obstruction-caused outlet resistance), bladder wall thickening
caused by matrix deposition and bSMC hypertrophy can lead to severely impaired renal
function15.
Since the mechanoreceptors embedded in the bladder wall sends afferent signaling to the CNS,
alterations in the bladder wall compliance can change bladder sensation and lead to inappropriate
activation of the bladder. In patients, obstructive uropathy and lowered bladder compliance are
often predicative for bladder overactivity.
1.2.3 Incomplete anatomical and functional reversal even after intervention
Clinically, bladder obstruction (anatomical and neurological) often arises insidiously and its
onset is not discovered until damage to the bladder is already evident. Through changes in
BSMC phenotype and bladder wall architecture remodeling, the bladder becomes hypertrophied
as well as distended (increased lumen size). Functionally, the bladder suffers from decreased
compliance and the inability to contract efficiently. Obstructive uropathy can lead to secondary
conditions such as incontinence, bladder overactivity, recurrent urinary tract infection and even
kidney failure. As a major urological complication, bladder obstruction compromises quality of
life and its associated societal cost is enormous. The updated cost of bladder dysfunction in the
U.S. in 2007 was $65 billion (with Canada estimates 10% of the cost) and the cost projections
are $76.2 billion and $82.6 billion, respectively by 2015 and 202017. Unfortunately, up to 40% of
the bladder obstruction patients do not regain normal bladder phenotype and functions even after
5
the removal of obstruction. Some patients continue to suffer from muscle wall hypertrophy,
altered organ compliance and diminished contractility after treatment18. Up to 23% of patients
with benign prostate hyperplasia continue to have abnormal bladder function parameters after the
removal of perturbing obstruction. Similarly, the bladder function in many patients with post
urethral valve failed to normalize even after many weeks of de-obstruction19. The mechanisms
underlying the persistent bladder pathology after removing the obstruction remains unknown.
Generally, the longer the duration of the bladder obstruction, the less potential there is for
complete anatomical and functional reversal. Clinically, however, it is very difficult to predict
the outcome of recovery for bladder obstruction patients. The goal of our study is to understand
the underlying mechanisms of bladder disease progression and mediator of the irreversible
pathology so that potential therapeutic targets can be identified.
1.2.4 Current treatments
Surgical de-obstruction and drug therapy is available to patients with obstructive uropathy.
Muscarinic anticholinergic drugs are the only available drug class to treat bladder obstruction
symptoms. Anticholinergic drugs work by inhibiting muscarinic cholinergic receptors, which
mediate efferent parasympathetic neurotransmitter signaling at the neuromuscular junctions20.
The anticholinergics can reduce BSMC contraction and bladder pressure. The anticholinergic
drugs, however, are ineffective in many patients. Alternatively, other drugs are used including
doxazosin (to improve sphincter relaxation 21), diazepam (GABA agonists to inhibit contractility
through central nervous system depression) and Botulinum toxin-A (inhibits reflex signaling
between neurons and bSMC 22), as previously reviewed1. Many of the recently surveyed clinical
trials on the treatment of bladder overactivity did not report efficacy23. More importantly, the
current pharmacotherapies only treat the symptoms and to date there is no drug treatment
available aimed to specifically correct the underlying muscle cell growth or the matrix
remodeling.
1.2.4.1 Rapamycin
Previously, our lab has shown that the mammalian target of rapamycin (mTOR) pathway
mediates BSMC proliferation and de-differentiation, induced by three inciting stimuli (stretch,
hypoxia and damaged matrix) associated with bladder obstruction (See Section 1.2.5 for
6
details)24. Downstream of the mTOR pathway are effectors (such as the p70 S6 Kinase, the
Autophagy related protein 13 and the eukaryotic Translation Initiation Factor 4G) that regulate
cell growth, proliferation, motility and survival 21. It is highly conserved across mammalian
species and it is regulated by many cellular signals such as stress conditions (strain and hypoxia),
growth factors and hormones 25.
Rapamycin inhibits the mTOR pathway by binding to the mTOR protein and inhibiting the
mTOR complex formation25. Our lab has shown that treatment with rapamycin in vitro inhibited
obstructive stimuli induced hyperproliferation and de-differentiation24. In rodent in vivo studies,
rapamycin prevented cardiac hypertrophy in response to mechanical strain26; and vascular SMC
proliferation in response to hypoxia 27.
In the present in vivo study (Chapter 3), we tested whether rapamycin can reverse changes
caused by established long-term obstruction after the release, which mimics the clinical setting as
bladder obstruction is usually diagnosed late (therefore damaged to bladder is already evident).
Previously, our lab reported that rapamycin treatment, given during the course of bladder
obstruction, was able to preserve bladder functional parameters and prevent muscle hypertrophy
as well as matrix remodeling28.
1.2.4.2 Incomplete reversibility in animal model of bladder obstruction
Bladder obstruction has been created in many animal models to better understand the progression
of pathophysiology and recovery after de-obstruction29-34. In these animal models, the
pathological progressions of obstruction are generally consistent with the disease in human
patients7. The bladder obstruction develops insidiously in animals without apparent morbidity.
Obstructed animals sacrificed at different timepoints shows a disease progression pattern
consistent with humans: the initial compensatory phase followed by the decompensatory phase7.
Similar physical hypertrophy and functional declines are also observed.
Furthermore, the phenomenon of incomplete bladder phenotype reversal is also observed in these
animal studies28-34. In a minipig study of bladder obstruction, many urodynamic parameters were
not reversed after de-obstruction followed by 3 months of recovery30. The de-obstructed bladders
still exhibited the lack of compliance, poor contractility (in isolated muscle) and a residual
7
volume that was three times as high compared to the non-obstructed control group. In another
study, roughly a third of the de-obstructed bladders had minimal functional improvement
(voiding pattern) 29 at the end of the recovery period and the sarcoplasmic reticulum Ca2+-
ATPase (SERCA) gene, which is important for calcium handling and therefore smooth muscle
function, remained dysregulated compared to the control.
The de-obstructed bladder often remains hypertrophic and less compliant. In a bladder
obstruction reversal study, after 7 weeks of obstruction plus 7 weeks of recovery after de-
obstruction, bladder weights were higher and the smooth muscle bundles were above control
values32. In another study, bladder weights decreased following recovery from obstruction but
remained higher than the control31. The level of BSMC hypertrophy, measured by smooth
muscle cell volume, was also decreased in obstruction followed by de-obstruction group
compared to the obstruction only group, but remained higher than control. In addition to
persistent hypertrophy, the ECM composition also remained altered following recovery from
bladder obstruction. The same study also reported that while the collagen fibrils in the control
group had a uniform diameter of 30 nm, the fibril diameter in the de-obstructed bladder was up
to twice as large with irregular orientations31. In another bladder obstruction study, the total
amount of collagen was increased three fold by obstruction and remained twice as high after 6
week of release33. Clearly there are ongoing defects with the BSMC phenotype, ECM and
compliance in bladders after the removal of obstruction, but a clear understanding of the
important features of the recovery process has yet to be elucidated.
Similar to human obstructive pathology, the bladder phenotype is less likely to revert in animals
with prolonged duration of obstruction. Therefore, by varying the duration of the obstruction we
can create reversible obstructions (short term, 2 weeks) and irreversible obstructions (long term,
6 weeks). Rat bladder that had undergone 2 weeks of obstruction, followed by 2 weeks of
obstruction relief, was able to resume a phenotype similar to that of the sham operated control
(Figure 1). On the other hand, bladder obstructed for 6 weeks remained hypertrophied and
distended even after 2 weeks of recovery from obstruction.
8
Figure 1 Duration of obstruction determines the degree of phenotype reversal following de-
obstruction
Bladder obstructions were created in female Sprague-Dawley rats for 2 weeks (short-term) and 6
weeks (long-term). The animals were allowed to recover for 2 weeks following surgical de-
obstruction. As shown, bladder was able to revert to a normal phenotype following short-term
obstruction and de-obstruction. However, bladder obstructed for 6 weeks failed to recover after 2
weeks of de-obstruction.
1.2.5 Three inciting stimuli during obstruction progression: stretch, hypoxia
and damaged matrix
During bladder obstruction, the overgrowth and partial loss of contractility is instigated by
coordinate responses to stimuli that causes BSMC to undergo phenotypic changes. As the urine
outflow is impeded, the excessive urine build-up causes bladder distention and wall tension. The
intramural pressure of the muscular wall increases proportionally to the size and volume of the
distended bladder, as stated by the Laplace’s law (tension in the wall of a hollow organ is
directly proportional to the radius of curvature.) Increased tension and pressure compresses the
microvasculature and creates tissue hypoxia35-37. Both stretch and hypoxia can lead to increased
metalloproteinase (MMP) activity and ECM remodeling38-41. ECM degradation releases
neoepitopes that induce BSMC proliferation and de-differentiation. Previously, our lab has
9
shown that MMP-7 activity is induced by hypoxia and the extracellular-regulated kinase
mitogen-activated protein kinase (ERK 1/2) mediates the hypoxia stimulated, MMP-7
transcription activation38. Using ex vivo rat bladders, we have also shown that distension
increases MMP activity as well as BSMC proliferation39. Together, these three stimuli cause
BSMC to undergo phenotypic changes such as hypertrophy, hyperproliferation and de-
differentiation. However, the mechanisms leading to this change are unknown and a better
understanding of SMC phenotypic switching is needed.
1.3 Smooth muscle cells
The smooth muscle cell is a major cell type in the body. Smooth muscle is generally categorized,
based on anatomical location, into vascular SMCs (lining the arteries, arterioles, veinules and
veins) and visceral SMCs (intestinal, gastric, urinary and reproductive systems). Vascular SMC
controls blood pressure and blood flow through blood vessel contraction or dilation. Visceral
SMCs are responsible for the contraction of organs such as the bladder, vas deferens, uterus,
ureter and the peristalsis of the digestive system42. Unlike skeletal muscle cells and
cardiomyocytes, SMCs appear “smooth” or non-striated under the microscope. Instead of
organizing the actin and myosin into the regular sarcomere pattern, a variable matrix of
contractile proteins inside the SMC is anchored to the dense body (analogous to the Z line of
striated muscle)42.
During muscle contraction, cytoskeletal intermediate filaments, that are attached to the dense
body, assist force transmission by harnessing the forces generated by the myosin crossbridge
activity2. To initiate a contraction, the rise in intracellular calcium level activates the myosin
light chain kinase; phosphorylated myosin light chains initiate the ATPase activity of the myosin
heads and thus leading to the formation of crossbridge contraction43 (as reviewed by 42). The
SMC is also more adaptable to changes in length than striated muscles. The luminal volume of
the bladder often expends substantially during filling (e.g. from approximately 0 mL to 400 mL).
The contractility and adaptability of SMC is altered by expression changes of SMC
differentiation markers during hypertrophy and/or de-differentiation in response to pathological
stimuli. This process is generally referred to as SMC phenotypic switching.
10
1.3.1 Smooth muscle plasticity
Unlike many other cell types, SMCs are highly plastic. In normal environment, differentiated
SMC tends to have a contractile phenotype characterized by a unique pattern of contractile
protein expression, low proliferation and protein synthesis. SMC can respond to the environment
and can transition into a more proliferative and synthetic state with fewer contractile protein
expressions. Li et al. has shown that SMC can readily shift between the two states44. When cells
were grown in vitro in the absence of fetal calf serum (FCS), SMC exhibited suppressed
proliferation, motility, ECM protein synthesis and mildly increased contractile protein expression
once the differentiated state was attained44. The characteristics associated with the differentiated
SMC phenotype were reversed under proliferative conditions (FCS treatment).
The differentiated SMC phenotype is attained by the expression of a unique set of genes, whose
encoded proteins coordinately achieve SMC related functional features. Many studies have
investigated the transcriptional regulation of SMC marker genes during the process of
differentiation and de-differentiation45,46. The majority of SMC-specific genes is regulated by
promoter element, an evolutionarily conserved DNA sequence called the CArG box
[CC(A/T)6GG] and the binding of serum response factor (SRF)45. The CArG DNA sequence is
present within 1-3 kb (kilo base pair) of the promoter or intronic regions of most SMC
differentiation markers 47,48. The SRF binds to CArG sequence as an homodimer and activates
gene expression related to muscle differentiation as well as proliferation49. The transcriptional
regulation of SMC phenotype is also regulated by myocardin (MYOCD), which is a potent co-
activator of SRF, that drives genes exclusively expressed in SMC as well as in cardiomyocytes49.
MYOCD and SRF binding in CArG elements is sufficient to achieve the contractile phenotype.
50 On the other hand, SRF can also bind to growth related transcription co-activators such as Elk-
1 and can regulate proliferation related genes47. Other SRF accessory factors include KLF4 51,
FoxO4 52 and SAP1 53. In response to pathological stimuli or changes in the microenvironment,
transcriptional repression of SMC related genes promotes the non-contractile phenotype54,55.
11
1.3.2 SMC phenotypic modulation and diseases
SMC phenotypic switching is considered to be an important underlying cause for smooth muscle
related diseases. In the development of atherosclerosis, lipid deposition alters the integrity of the
endothelium, which permits monocyte invasion into the SMC layer56. Growth factors and
inflammatory mediators are released into the microenvironment and causes SMC to transition
from the contractile phenotype to the de-differentiated/synthetic phenotype. The de-differentiated
SMCs then migrate into the intima and contribute to intima thickening 57,58 (reviewed by 59).
Aneurysms can also develop as a result of SMC phenotypic switching. Aortic SMCs transition
into a state with decreased expression of contractile proteins and increased secretion of MMPs.
This is followed by SMC apoptosis, further weakening of the blood vessel wall and ultimately
vessel rupture60. (reviewed by 59) During bladder obstruction, coordinate inciting stimuli (strain,
hypoxia and damaged matrix) cause phenotypic switching of BSMC (See section 1.2.5 for
details).
Through changes in gene expression60, SMC assumes a hyper-proliferative, hypertrophic and de-
differentiated state. Many studies have reported on changes in the expression profile of
contractile proteins, cell adhesion molecules and member receptors during SMC phenotypic
switching61-67. The differentiated state for SMC is usually marked by the expression of
contractile proteins such as ACTA2 (-SMA), SM22, h-calponin and h-caldesmon. During
obstruction, there is increased l-caldesmon expression compared to h-caldesmon isoform68,69.
The ratio of myosin heavy chain isoforms (SM2-to-SM1) is strongly correlated to increased
bladder weight post-obstruction60,70.
The protein level of -SMA is positively correlated to the degree of differentiation in SMC
grown in culture44. Heat denaturation has been used as an in vitro substitute for ECM
degradation in vivo71. Moreover, the study by Jones et al. showed that damaged matrix induces
upregulation of tenascin-C71, an extracellular glycoprotein that alters vascular SMC morphology
and amplifies proliferative responses72.
In the past, our lab has also shown that BSMCs are highly responsive to changes in the
microenvironment40. Primary rat BSMCs cultured on normal type I collagen had spindle-shaped
12
appearance while cells grown on heat denatured collagen showed greater cell spreading with
more stress fibers. In addition to profound morphological changes, damaged collagen also
induced significant level of BSMC hyperproliferation40. Therefore, the extracellular matrix
environment is an important modulator of SMC phenotype.
1.4 The extracellular matrix
The extracellular matrix provides a foundation basis for multicellularity and it provides structural
support to many organs such as the skeletal system, the vasculature and hollow organs. It is a
scaffold comprised of fibrillar proteins, proteoglycans and glycosaminoglycans (GAGs)73. In
addition to structural support, the ECM also acts as a growth factor reservoir and ECM
degradation can release growth factors that induce proliferation. Therefore, by varying the degree
of stiffness/compliance and by the storage/ release of growth factors, the ECM provides
signaling cues to the residential cells and modulates cell behavior during development and
disease.
1.4.1 Matrix and cell phenotype regulation
The extracellular matrix is not an inert scaffold. It is continuously remodeled by enzymatic
digestion and crosslinking. For example, lysyl oxidase crosslinks fibrillar collagen and elastins
and MMPs enzymatically breaks down various components of the ECM. Through remodeling,
the matrix stores or releases as well as modulates the activity of growth factors, cytokines and
other proteases73-75. These growth factors signal cells to undergo phenotypic changes via
signaling pathways. Alternatively, changes in ECM architecture alter the compliance and
stiffness of the matrix and this mechanical signal is transmitted to the nucleus by the focal
adhesion molecules and subsequent cytoskeletal rearrangement 76,77 (reviewed by 78). Through
these two processes, changes in the ECM affects gene expression and subsequently affects
properties of the cell. In turn, changes in gene expression can alter the amount as well as
bioactivity of matrix remodeling enzymes, leading to further interactions between the residential
cell and the metrical environment. The dynamic and two-way communication between the cell
and the matrix, is known as dynamic reciprocity79. Different components of the matrix can
13
influence many pathways either indirectly (intracellular transduction pathway) or directly
(cytoskeleton reorganization) and can lead cells to different fates such as proliferation, apoptosis
and differentiation.
Indeed, the ECM can induce drastic changes in cell phenotype, even in cells that are considered
to be terminally differentiated. For example, mammary epithelia, upon clearing the epithelia by
transplantation upon mammary fat pads, formed the entire mammary epithelial tree80 (reviewed
by 81). Murine ectodermal cells from adult seminiferous tubules underwent phenotypic switching
to exhibit mammary epithelial behavior when mixed with mammary epithelial cells and placed in
epithelia-free fat pads82 ( reviewed by 81). Therefore, cell identity is dependent on its surrounding
microenvironment. In fact, Bissell proposed that the differentiated state of adult cells is not truly
terminal, but rather “contextual of its organ microenvironment”. 81 It follows that during the
maintenance of homeostasis, the extracellular matrices from different organs keep the residential
cells in their appropriate differentiated states by preventing abnormal growth ( 83,84 reviewed by
81) and phenotypic switching81. In cancer studies, it is well known that ECM components,
architecture and the expression, as well as activity of matrix modifying enzymes, are key
regulators of cancer cell invasion and metastasis85. Similarly, these ECM characteristics are also
remodeled during non-malignant diseases and in turn modulate the pathological progressions.
1.4.2 The extracellular matrix of the normal and obstructed bladder
As mentioned above, the physical and mechanical properties of the bladder wall are critical for
the proper filling and voiding functions. During bladder obstruction, the architecture and
components of the bladder extracellular matrix are modified and this leads to changes in the
physical properties of the muscular wall.
The bulk of the bladder matrix is comprised of fibrillar collagen I and collagen III. Collagen
fibers are comprised of a triple helix of three -polypeptide chains with the repeating sequence
of Gly-x-y86. Collagen I is mainly located in the lamina propria and the matrix surrounding
muscle bundles and it provides structure support and tensile strength87. Proper collagen I
deposition is critical for bladder function88 (reviewed by 89). Collagen III is integral for the
correct assembly of collagen I fibers90. Elastic fibers are composed of tropoelastin embedded in
14
microfibrillar proteins 91,92 (reviewed by 89). Elastins are responsible for the elastic properties that
allow the bladder to recoil back to its original shape after urine expulsion. The interplay between
the ECM and cells is mediated by ECM receptors such as the integrins. They transduce
biochemical signals and physio-mechanical information from the matrix to the cytoplasm45.
Under normal physiological conditions, the maintenance of matrix homeostasis is achieved by
regulated activities of the matrix crosslinking proteins and the matrix degradation proteins.
During bladder obstruction, however, the composition and the integrity of the bladder wall ECM
is altered. Firstly, many studies have reported the increased deposition of fibrillar collagen III93-97
(reviewed by 89). Chang et al., showed that type III collagen changes three dimensional
conformations at different stages of bladder filling and that these conformational changes
contribute to the compliant properties of the bladder wall 87. During the process of bladder wall
hypertrophy, however, the function of type III collagen may be altered as studies have found that
increased expression of collagen III contributes to decrease wall compliance 98,99.
In addition to altered ECM composition, bladder obstruction also upregulates MMP activities.
Both in vivo bladder obstruction studies as well as in vitro BSMC strain studies report the
induction of MMP2 100 , MMP7 24, MMP9 and MMP28 101(reviewed by 89). Enzymatic
breakdown of the matrix can release cryptic neoepitopes that change the smooth muscle behavior
through the activation of intracellular signaling pathways.
1.4.3 Matrix regulation of smooth muscle phenotype
During obstructive uropathy, damaged matrix, either broken down by the excessive pressure of
the bladder wall or by proteolytic enzymes, can exacerbate stretch induced injury and cause
BSMC phenotype changes. Our lab has shown that conditioned media from ex vivo bladder
distention treatment can cause proteolytic breakdown of type I collagen by MMP2 and release
mitogenic factors39. In another study, heat denatured type I collagen caused BSMC to undergo
proliferation and de-differentiation40. In vascular diseases, the integrin v3 is activated by
MMP activity and mediates vascular SMC migration as well as proliferation on denatured
collagen66,71. In strain-induced injury, integrin signaling is also an important mediator of bladder
SMC proliferation102. Interestingly, the damaged matrix induced mitogenicity is only partially
15
reversible in vitro40. When the hyper-proliferative BSMCs were released from damaged type I
collagen matrix and passaged back onto normal collagen matrix (thus removing the pathological
stimulus), only a partial normalization occurred in the proliferation rate. The incomplete
reversibility of the hyper-proliferative and de-differentiated phenotype, even upon the return to
normal matrix, suggests that ECM induced phenotypic switching could be mediated by
epigenetic mechanisms.
1.5 Epigenetics
Epigenetic changes refer to modifications to DNA or the chromatin without changing the DNA
sequence103. Major mechanisms of epigenetic modifications include DNA methylation and
histone modification, both of which can regulate gene expression by affecting the DNA
accessibility to various DNA binding proteins (reviewed by Duncan et al.103). In recent years,
non-coding RNAs such as long non-coding RNA (lncRNA), microRNA and piwiRNA have been
recognized as the third type of epigenetic regulation as they can regulate mRNA expression can
interact with both DNA methylation and histone modification104. Together with transcription
factor signaling, these three mechanisms set the tissue specific epigenetic and expression
landscapes in cells and regulate differentiation of different cell types during development. Since
epigenetic regulation machinery controls cell differentiation fate and is influenced by the
environment105,106, the epigenetic landscape can be viewed as a dynamic interface between
environmental stimuli and the genome. Changes in epigenetic machinery in response to the
environment can alter cell phenotype and may provide a means for the organism to adapt. In
pathological progression, however, this key interface may change gene expression profiles in
ways that result in persistently abnormal cell behaviors.
1.5.1 Mechanisms of epigenetic regulation
The DNA packaging nucleosome consists of a segment of DNA and eight histone proteins,
which is comprised of 2 copies of each core histone H2A, H2B, H3 and H4107. Various post-
16
translational histone modifications, such as acetylation, methylation and phosphorylation 108 can
be catalyzed by enzymes to control DNA packaging. Histone modifying enzymes can, therefore,
regulate gene expression by manipulating the degree of DNA accessibility to transcription
factors109,110. On the other hand, non-coding RNA is a class of functional RNAs that are not
translated into a protein. Non-coding RNAs can target histone modifications at particular loci
111,112 and downregulate gene expression by inducing complementary RNA degradation113.
1.5.1.1 DNA methylation
In mammals, DNA methylation involves transferring a methyl group onto the fifth carbon of the
cytosine residues; this covalent modification is catalyzed by a class of enzymes called DNA
methyltransferases (DNMTs) 114 (reviewed by Jurkowska et al.115 ). DNA methylation occurs
usually on CG dinucleotides (refered as CpG) but non-CG methylation can also occur. A
sequence longer than 550 basepairs, concentrated with CpG dinucleotides is referred to as CpG
islands116,117 and they are found in the promoter regions of roughly 70% of human genes
(reviewed by Jurkowska et al.113). DNA hypermethylation can influence transcription factor
binding and reduce gene expression or induce gene imprinting. Alternatively, methylation of
DNA in non-promoter regions can influence splice variants. There are two types of DNMTs.
DNMT1 is referred to as the “maintenance” enzyme because it has a preference for
hemimethylated DNA and is responsible for maintaining the DNA methylation marks on the
newly synthesized strand of DNA during cell replication. DNMT3A and DNMT3B are referred
to as the de novo methyltranferases 118 because they can establish new methylation marks in
response to cell signaling pathways. DNMT3L is a non-catalytic variant that acts as key co-
factor to DNMT3A and DNMT3B119.
Mechanisms that regulate catalytic activity and targeting of the de novo DNMTs are multifold
and interconnected. Two isoforms of DNMT3A and six major isoforms of DNMT3B have been
described (reviewed by Choi et al. 120). Their subcellular localization, stability and catalytic
activities are regulated by many signaling pathways. In a study by Choi et al., parallel Illumina
DNA methylation assays were performed on 11 HEK 293T cell lines stably expressing
exogenous DNMT3 isoforms to examine their downstream targets. The transfection of major
isoforms of DNMT3A and DNMT3B induced hypermethylation of CpG dinecleotides in both
CpG islands and non-CpG islands, suggest diverse functional roles of the de novo DNMTs.
17
There appear to be high degrees of overlap between downstream targets of 3A isoforms and 3B
isoforms. This claim, however, can be misleading and the degree of overlap between DNMT
variants is likely exaggerated due to the very limited number of surveyed CpG sites. Only 1505
sites from 808 genes were surveyed in the study and the total number of CpG sites in the human
genome is estimated at 56 million121. The degree of overlap of downstream targets between the
isoforms, therefore, cannot be evaluated based on surveying such a small set of genes.
Interestingly, DNMT3A1 and DNMT3B1 almost exclusively correlated with H3K4me3
(transcription activation) and H3K27me3 (transcription repression), respectively. The two
variants may selectively target loci that are previously active and inactive by the association of
their unique structural domains with different histone modifications.
1.5.2 Epigenetic modulation of SMC phenotype
Studies have shown that SMC phenotypic modulation during vascular and bladder disease
progressions involved epigenetic mediators122. For example, Platelet derived growth factor
(PDGF)-BB induced vascular SMC phenotypic switching is facilitated by the compaction of
chromatin at SMC differentiation marker genes123. In response to PDGF-BB induction, KLF4
recruited histone deacetylases (HDACs) to CArG sequences of ACTA2 gene and inhibited
transcription activator (SRF and MYOCD) binding124. In another study, excessive collagen
production in vitro by BSMC, isolated from patients with neurogenic bladders (vs. healthy
bladders), is decreased by the treatment of HDAC inhibitor trichostatin A (TSA) 125. In another
murine bladder obstruction study, analysis of mRNA microarray showed that decreased
expression of miR-29 following bladder obstruction was associated with increased levels of miR-
29 target genes (e.g. collagen IV) 126.
A few other studies suggest that matrix regulation of SMC phenotype is mediated by epigenetic
mechanisms127-129 ( reviewed by 59). Collagen XV gene is hypomethylated during SMC de-
differentiation and the DNA methylation inhibitor 5-Aza-2'-deoxycytidine (DAC) was able to
prevent the increased expression of collagen XV and the SMC proliferative phenotype127. In a
study investigating primary rat airway SMC differentiation, DAC treatment inhibited PDGF-
induced cell proliferation and cytoskeletal re-organization while improving the SMC
contractility128. A study using patient samples found that MMP1 was hypomethylated in the
omental arteries of preeclamptic women and DAC treatment stimulated the protein secretion of
MMP1 in vascular SMC in vitro129.
18
1.6 Hypothesis and Aims
The persistence of pathology (hyper-proliferation, hypertrophy and de-differentiation) in humans,
animals as well as cells in vitro after the removal of inciting stimuli (strain, damaged matrix and
hypoxia), suggests that the epigenetic machinery may be involved in the progression of partial
bladder outlet obstruction. Many studies have reported on cardiomyocyte and vascular SMC
phenotypic modulation by epigenetic machinery (mainly by histone modifications)123-126,130-136.
The research on how DNA methylation machinery mediates BSMC phenotypic change during
bladder obstruction is scarce despite the fact that DNA methylation is considered the more stable
epigenetic alteration and the well-known pathological irreversibility associated with long-term
bladder obstruction.
1.6.1 Hypothesis
We hypothesized that DNA methylation changes mediate bladder smooth muscle cell
phenotypic switching during bladder obstruction.
1.6.2 Specific aims and experimental plans
Specific Aim 1: Examine the role of epigenetic machinery mediating aberrant ECM induced
BSMC phenotypic changes.
Previously, we observed that the aberrant matrix microenvironment alone can incite a stable
phenotype alteration in BSMC, which is not completely reversed upon the return to normal
matrix40. The DNA methylation machinery of BSMC will be examined using an in vitro model
of damaged ECM (an inciting stimulus during bladder obstruction). Previously, other studies
19
have used heat denatured type I collagen as an in vitro surrogate to study SMC phenotypic
switching and gene expression changes in response to the damaged ECM in vivo 40, 71.
1. The DNA methylation machinery, specifically the expression and localization of the DNMT
proteins, will be examined in BSMC plated on NC or DNC by immunofluorescence and
western blot experiments.
2. To elucidate epigenetic signaling effectors in irreversible bladder obstruction that mediate
persistent BSMC growth in vitro, BSMC cultured on aberrant ECM will be treated with
pharmacological signaling pathway inhibitors.
3. Lastly, DNA methylation changes, induced by damaged matrix, will be examined.
Specific Aim 2: Determine if DNA methylation changes occur during bladder obstruction.
Longer duration of bladder obstruction decreases the potential for bladder phenotype reversal
following the relief of obstruction. The experimental animal bladder, therefore, provides a
unique platform to create a model of irreversible obstructive bladder disease. Aim 2 will reveal
whether long-term bladder obstruction leads to persistent gene dysregulation and/or DNA
methylation changes.
1. Bladder obstruction and PCR array: Using female rats, different bladder obstruction groups,
including long-term bladder obstruction (6 weeks), long-term bladder obstruction followed
by release (6 weeks plus 6 weeks release) and sham operated control (12 weeks) will be
created.
2. The expression profile of eighty-eight SMC differentiation related genes (curated from
bladder obstruction literature) will be compared using a Qiagen custom PCR array.
3. Subsequently, the methylation states of persistently dysregulated genes (following the relief
of obstruction) will be compared across different treatment groups.
20
2 Chapter 2: Smooth Muscle Cell Phenotypic Switching
Induced by Damaged Matrix Is Associated with Changes in
DNA Methylation
2.1 Methods
2.1.1 Bladder Smooth Muscle Cell (BSMC) culture
“Neonatal [rat] pups (postnatal days 1-3) were housed under normal light/dark conditions with
their dam (free access to food and water) until removed from the cage and sacrificed by
decapitation in accordance with approved protocol with the Animal Care Committee of the
Hospital for Sick Children.” Bladders were harvested, minced and then digested with type IV
collagenase (2 mg/mL) for 5 minutes (Sigma-Aldrich). Suspended cells were removed from the
final digest, and tissue was further digested for another 40 minutes.” (directly quoted from 137)
An average of 2 bladders were grown on a 10 cm dish (BD Falcon) for 1- 2 weeks in 10% fetal
bovine serum (FBS, Multicell) in EMEM with the appropriate antibiotic/antimycotic drugs
(Multicell). Upon 90% confluency, primary rat BSMCs were released from the plate using
0.25% trypsin/EDTA (Multicell) as per manufacturer’s protocol and subcultured. Cells were
subcultured for up to 2 passages before used in experiments.
Purchased primary human BSMCs (PromoCell and ScienCell) were maintained in Smooth
Muscle Cell Medium (ScienCell) and passaged using 0.25% trypsin/EDTA (Multicell) as per
manufacturer’s protocols. Cells were subcultured for up to 6 passages before use in experiments.
Both human and rat BSMCs were starved in 0% FBS EMEM for 24 hours before use in
experiments.
2.1.2 Primary huBSMC transfection
The plasmid pcDNA3/Myc-DNMT3A was a gift from Arthur Riggs (Addgene plasmid #
35521)119. Single colonies were selected from the agar plates and the bacteria was inoculated in
21
liquid Luria broth (LB) overnight with agitation. The plasmid was isolated using the Miniprep
kit (Qiagen). Isolated plasmid was enzyme digested and verified by the band size on an agarose
gel as well as DNA sequencing.
Purchased primary huBSMC was transfected with the myc-DNMT3A plasmid using
Lipofectamine 1000 (Life Technologies) or the Nucleofector II (Lonza) system according to
manufacturers’ protocols. (See Appendix I for detail)
2.1.3 Preparation of Collagen Substrates
Two substrates of collagen were prepared using type I collagen (Elastin Products Company,
Owensville, Missouri). The normal/native collagen was prepared by mixing equal volumes of 6
mg/mL collagen and a 0.1 M NaOH + 2X PBS solution. NC matrix was polymerized under 37°
C for 1 hour. The denatured/damaged collagen (DNC) was first boiled for 20 minutes and then
neutralized using the same basic solution. Both NC and DNC were layered onto glass coverslips
in 24 well plates (BD Falcon), or directly onto 6 well plates and 10 cm dishes for
immunostaining, RNA/DNA extraction or protein extraction experiments. NC and DNC were
washed with EMEM three times before use in experiments. BSMCs were plated at 5 x 104
cells/mL, unless otherwise stated. For Figure 2, cells were plated onto NC alone, a 1:2 mixture of
NC and DNC or DNC alone.
2.1.4 Hypoxia
Serum starved rat BSMCs were plated onto NC/DNC matrices, incubated either under normoxic
conditions at 21% O2 or in a humidified hypoxia chamber (Biospherix Pro-Ox 110 Oxygen
Controller, New York) at 3% O2, 5% CO2 and 92% N2 for 16 hours.
2.1.5 Immunostaining and Confocal Microscopy
For immunofluorescence experiments, cells were fixed with 4% paraformaldehyde (PFA) for 20
minutes (followed by 3 PBS washes,) permeabilized in 0.2% Triton X-100 in PBS (followed by
3 PBS washes) and blocked by 3% BSA plus 10% normal goat or donkey serum. The stainings
were performed with the appropriate primary antibody at 4°C for one hour or overnight
(followed by 3 PBS washes) and the corresponding secondary antibodies diluted to 1:200 for one
22
hour. See Table 1 for a full list of primary and secondary antibodies used. “The nucleus
counterstaining was performed with Hoechst (Life Technologies, 1:1000 dilution in PBS, 5
minutes followed by PBS wash).” (directly quoted from 137) Cells were then mounted and sealed
with mounting medium (Dako) and visualized using confocal microscope and the Velocity
software. The intensity of fluorescent signal was analyzed and quantified using ImageJ.
Table 1 List of immunofluorescence antibodies
1°/2° Antibody Species Dilution Company
1 myosin polyclonal rabbit 1/200 Abcam
1 DNMT3A monoclonal mouse,
polyclonal rabbit
1/200 Abcam
1 SMA polyclonal rabbit 1/200 Abcam
1 Myc-tag polyclonal rabbit 1/200 Abcam
1 Myc-tag monoclonal mouse 1/300 Cell Signaling
2 Green ,cy2 rabbit 1/200 Jackson Immunolabs
2 Red, cy3 mouse 1/200 Jackson Immunolabs
2 Far-Red rabbit 1/200 Jackson Immunolabs
2.1.6 Cell Counting
For evaluations of proliferation and survival, bromodeoxyuridine (BrdU) staining was not used
in cell counting experiments because the hydrochloric acid treatment with the staining protocol
solubilizes collagen gels. Instead Hoechst stained nuclei were visualized by Velocity and
counted using ImageJ on a minimum of 9 fields (200X magnification).
2.1.7 Protein Extraction methods and Western blotting
BSMCs were released from matrices using either collagenase (2mg/mL, 5 minute incubation at
37°C) or trypsin/EDTA (0.25%, 5 minutes incubation at 37°C) digestion. Cells were lysed by
suspension in 0.5% deoxycholate in Tris buffer with a protease inhibitor mix (Invitrogen). After
10 minutes of incubation on ice, the lysate was centrifuged at 10,000 g at 4°C for 10 minutes.
The protein containing supernatant was transferred into a new tube. The amount of protein was
23
quantified with a BCA Protein Assay Reagent (Thermo Scientific) and 20 ug of each protein
sample was diluted in Laemmli sample buffer and heat denatured. “Protein was electrophoresed
on an 8% PAGE gel, transferred to nitrocellulose membrane via electroblotting.” (directly quoted
from 137) Membranes were blocked in 5% BSA and 5% skim milk powder in TBST, and probed
with various antibodies overnight at 4°C with agitation. See Table 2 for a full list of antibodies
used. Secondary anti-mouse- or anti-rabbit-HRP (1:1000) and ECL-Plus were used to detect
bands via autoradiography.
Table 2 List of western antibodies
Antibody Species Dilution Company
DNMT3A polyclonal rabbit 1/1000 Abcam
Gapdh polyclonal rabbit 1/1000 Cell Signaling
Myc-tag monoclonal mouse 1/1000 Cell Signaling
2.1.8 RNA extraction, Reverse Transcription and Polymerase Chain Reaction
(PCR)
RNA from rat or human BSMCs was isolated using Trizol (Invitrogen) as per manufacturer’s
protocol and quantified with NanoDrop 2000 (Thermo Scientific). Up to 1.0 ug of RNA was
reverse transcribed using either the Superscript III (Invitrogen) or the RT2 First Strand Kit
(Qiagen). “PCR was performed on the Peltier Thermal Cycler-100 (MJ Research) with the iQ
SyBR Green mix (BioRad) or the RT2 SYBR Green Master Mixes (Qiagen). Quantification was
done using the delta-delta cT method.” (directly quoted from 137)
2.1.9 Agarose gel electrophoresis
1.5 g of agarose was dissolved into 100 mL of TAE buffer by boiling and was allowed to cool
down before the addition of 5 uL of the RedSafe (JH Science). The agarose was poured into the
gel tray and allowed to sit until completely solidified. The solidified gel was transferred into the
gel box and completely covered with TAE buffer. The DNA samples were mixed with the
24
appropriate volume of the 6x loading buffer (Thermo Scientific) and loaded into the gel along
with the GeneRuler DNA Ladder (Thermo Scientific). The gel was run at 90 volts until the
sample dye had migrated approximately 80% down the entire gel. The gel was visualized using a
Gel Doc XR+ System (Bio-Rad).
2.1.10 Drugs and treatments
Drugs, inhibitors and growth factors were applied to cells 3-6 hours after the initial plating on
different matrices to avoid potential interference with cell attachment and spreading. The drugs
were re-applied in fresh medium according to their half-life duration. See Table 3 for a full list of
drug treatments and concentrations.
Table 3 List of drug treatments
Treatment Dilution Company
Rapamycin 5 ng/mL LC Laboratories
DAC 0.2, 1 ,3 uM Sigma-Aldrich
Cyclohexamide 10 ug/mL LC Laboratories
Actinomycin 0.5 ug/mL Sigma-Aldrich
Nocodazole 0.04 ug/mL Sigma-Aldrich
EGF 50 ug/mL BD Transduction
bFGF 10 ug/mL BD Transduction
PD98059 40 uM Sigma-Aldrich
F11 0.03 uM Sigma-Aldrich
Leptomycin B 20 ug/mL Cell Signaling
2.1.11 Illumina Bead-chip analysis of DNA methylation on damaged matrix
“Primary culture human bladder smooth muscle cells (Obtained from PromoCell), were cultured
on normal collagen and denatured collagen for 48 hours in vitro. DNA was extracted and
bisulfite converted using the EZ DNA Methylation-Gold kit (Zymo Research), and then
amplified by Illumina Infinium HD Methylation assay and hybridized to a Human 450 K
methylation v1 Beadchip. The Beadchip was scanned using iScan (Illumina) and quantified in
25
GenomeStudio Version 2011.1 (Illumina). Microarray service was provided by the University
Health Network Princess Margaret Genomic Centre (www.pmgenomics.ca Toronto, Canada).”
(directly quoted from 137)
2.1.12 Statistics
Unless otherwise indicated, experiments were conducted with the sample size of at least n= 4 in
each treatment group. “Comparisons between groups were performed using an analysis of
variance or a two-tailed t-test. A p value less than 0.05 was considered significant. For
MethylArray data, statistical analysis was performed on R Bioconductor IMA and Methylumi
packages, for both the Welch’s t-test and adjusted p values. For the total epigenomic analysis, a
Benjamini-Hochberg correction for multiple testing was performed on data, p less than 0.05 and
t-test less than 0.01. For the a priori analysis, we analyzed CpG sites proximal to SMC
differentiation related genes (as identified on Ingenuity pathway analysis). This set of CpG sites
(6831 sites) were analyzed separately from the main list, using Benjamini-Hochberg to correct
for multiple testing, after which p less than 0.05 was considered significant.” (directly quoted
from 137)
26
2.2 Results
2.2.1 Damaged Matrix-induced cell proliferation and de-differentiation is
dependent on DNMT activity
Previously our lab has shown that smooth muscle cells show accelerated proliferation when
cultured on damaged collagen matrix (DNC) compared to normal collagen matrix (NC). In order
to test whether this DNC-induced hyper-proliferation is mediated by the DNA methylation
machinery, we used the DNA methylation inhibitor, 5’-aza-2’-deoxycytidine (DAC). Consistent
with our previous findings, damaged matrix induced significant BSMC hyperproliferation at 48
hours after plating while normal matrix rendered BSMC quiescent (Figure 2 A). A decreased
expression of myosin, an important SMC differentiation marker, is also observed on DNC
(Figure 2 B). DAC treatment attenuated DNC-induced hyper-proliferation as it prevented the
increase in cell number on damaged matrix without affecting the cell numbers on normal matrix.
The inhibition of DNA methylation prevents the hyper-proliferation on DNC without affecting
the basal proliferation rate on NC, suggesting that hyper-proliferation of BSMC on DNC
depends on DNMT activity.
“Previous experiments showed that rapamycin can prevent the loss of myosin on denatured
collagen. 40 However, recovery of myosin after prior culture on DNC, was only seen by
combining rapamycin with epigenetic inhibitor treatment (DAC) (Figure 1 B).” (directly quoted
from 137)
27
Figure 2: Proliferation and de-differentiation of SMC on denatured matrix depends on DNMT
activity in visceral smooth muscle cells.
SMC were plated on native (NC) or denatured collagen (DNC) at low density (2×104
cells/mL) for 6
hours in EMEM with 6% FCS, then treated with 5-aza-2′-deoxycytidine (DAC) or vehicle for another 42
hours in EMEM with 2% FCS. Six different fields per treatment for cells positive for DAPI were
examined at 5× magnification and counted using Volocity analysis software, and averaged to obtain the #
cells/field. * p<0.05 (n=4) vs DAC treatments. (B) Loss of smooth muscle myosin could be reversed with
rapamycin plus DAC. Before treatment, SMC were cultured for 48 hours in vitro on damaged collagen
matrices (DNC), which suppressed expression of the differentiation marker Myosin (relative
immunofluorescence expression = 1.0). The mTOR inhibitor rapamycin alone showed only a trend in
increasing Myosin expression (p = 0.11, n=4), but combined use of epigenetic inhibition (with DAC) +
rapamycin significantly restored myosin expression (*p<0.04, n=4).
28
2.2.2 Matrix alters intracellular DNA methyltransferase 3A (DNMT3A)
localization and expression in visceral smooth muscle cells
Since the inhibition of DNA methylation was able to prevent BSMC hyper-proliferation on the
damaged collagen, we investigated whether DNMT3A is regulated by matrix. At 48 hours after
plating, the nuclear expression of DNMT3A was profoundly increased in cells cultured on DNC
compared to the basal and cytosolic DNMT3A expression in cells on NC (Figure 3 A).
Increasing the proportion of denatured collagen from a NC-DNC mixture (1:1 ratio) to DNC
resulted in an increase in DNMT3A nuclear expression. It is worth noting that the change in
DNMT3A subcellular localization of DNC is especially noteworthy as others have also shown
that increased DNMT nuclear expression strongly indicates an epigenetic response because DNA
modification occurs in the nucleus. Furthermore, the change in DNMT3A localization was
accompanied by a decrease in the level of -smooth muscle actin (-SMA), an important SMC
differentiation marker.
We also examined whether DNC changes the total level of DNMT3A expression (Figure 3 B).
With Western blot, we observed a clear increase in DNMT3A protein expression in cells plated
on DNC at 48 hours.
2.2.3 Hypoxia potentiates nuclear DNMT3A expression on DNC
During bladder obstruction, hypoxia acts as a co-stimulus that induces BSMCs to undergo
hyperproliferation and de-differentiation. We have shown that hypoxia induces visceral SMC
proliferation24,38 and Watson et al. demonstrated that hypoxia can alter the DNA methylation
machinery during cardiac cell phenotypic switching138. We were interested in whether hypoxia
alters the DNMT3A expression or localization upon exposure to damaged matrix. Primary rat
BSMCs were plated on NC or DNC and cultured under normoxia (21% O2) or hypoxia (3% O2).
On damaged matrix, hypoxia profoundly potentiated the nuclear upregulation of DNMT3A at 48
hours (Figure 4 A) while exacerbating the loss of differentiation, as observed by the
immunostaining of DNMT3A and myosin. Saturation of fluorescence occurred for the hypoxia
plus DNC group under regular microscope exposure used to observe cells on DNC alone.
29
Furthermore, the mRNA expression of SMA was reduced and the expression of DNMT3A
was significantly enhanced by hypoxia in addition to damaged matrix. (Figure 4 B)
Figure 3: Matrix is a critical determinant of DNMT3A expression in visceral smooth muscle cells.
SMC were plated on native (NC) or denatured collagen (DNC) at low density (4×104 cells/mL) for 6
hours in EMEM with 6% FCS, then media was changed to 2% FCS in EMEM. (A) DNMT3A expression
increases in the nucleus in response to denatured matrix, while α-smooth muscle actin (α-SMA)
expression decreased. By immunofluorescent staining, levels of DNMT3A and SMA were examined with
spinning disk microscopy using Volocity software, then analysed with Image J. *, p<0.05, n=4. (B)
Western blotting of DNMT3A1 in protein extracts isolated from rat bSMC cultured on NC and DNC.
Damaged matrix induced higher protein expression of DNMT3A1 (120 kDa).
30
Figure 4: Hypoxia and damaged matrix increase DNMT3A nuclear expression in a cooperative
fashion.
SMCs were plated on native (NC) or denatured collagen (DNC) at low density (4×104 cells/mL) for 6
hours in EMEM with 6% FCS, then media was changed to 2% FCS in EMEM. (A) SMC were plated on
native (NC) or denatured collagen (DNC) and cultured under normoxia (21% O2) or hypoxia (3% O2).
Hypoxia significantly enhanced the nuclear expression of DNMT3A and the down-regulation of myosin.
By immunofluorescent staining, levels of DNMT3A and smooth muscle myosin heavy chain (MHC,
smooth muscle-specific form) were examined by spinning disk microscopy using Volocity software, then
analysed with Image J. *, p<0.05, n=4. (B) Expression of α-SMA was significantly decreased under the
combined stimulation by hypoxia and damaged collagen, compared to native collagen. Both PCR and
immunofluorescent staining with anti-smooth muscle actin antibody revealed a significant decrease in
actin expression only on denatured collagen. (C) The expression of DNMT3A is upregulated in DNC
compared to NC. Consistent with immunofluorescent staining data, the upregulation of DNMT3A mRNA
expression on DNC is enhanced by hypoxia (n=4).
31
2.2.4 Matrix regulation of DNMT3A depends upon culture duration,
transcription and translation
We investigated if the nuclear localization of DNMT3A on damaged matrix is dependent on
culture duration and transcriptional as well as translational regulation as a cell’s response to
changes in the microenvironment may require all three. First, a timecourse of BSMCs cultured
on different matrices was conducted where cells were cultured on NC or DNC and fixed for
immunostaining at 6, 12, 24, 36 and 48 hours after plating. Consistent with our previous data,
DNMT3A nuclear localization occurred at 48 hours. The intracellular expression of DNMT3A at
earlier timepoints showed that the nuclear translocation started to occur as early as 6 hours on
DNC (Figure 5 A).
Moreover, “we examined whether transcription and translation are required for nuclear
upregulation of DNMT3A using chemical inhibitors of these processes. Inhibition of
transcription by cyclohexamide downregulated DNMT3A nuclear expression (Figure 5 B).
Actinomycin D appeared to have only a mild, if any, effect on DNMT3A localization, though it
appears that both transcription and translation are required for the downregulation of SMA on
DNC.” (directly quoted from 137)
2.2.5 Increased DNMT3A expression does not alter its localization
Since DNC increased total protein expression of DNMT3A, the DNC-induced nuclear DNMT3A
upregulation could result from greater amounts of the protein available for nuclear import. To
examine if the DNMT3A protein expression regulates its localization, I transfected primary
human BSMCs with myc-tagged DNMT3A and then plated the cells on different matrices. As
shown in Figure 6, increased level of DNMT3A by transfection does not alter the localization of
this protein. Consistent with non-transfected cells, both the endogenous and the myc-tagged
DNMT3A is cytosolic on NC, nuclear on DNC at 48 hours. This suggests that the extracellular
matrix has a predominant effect on DNMT3A’s subcellular localization.
32
Figure 5: Nuclear Localization of DNMT3A is dependent upon the time after plating and
transcription.
(A) Timecourse of intracellular DNMT3A expression/localization after plating cells on NC and DNC
(n=4). DNC plated cells show stronger DNMT3A signals overall than NC plated cells. The 36 hour
timepoint shows strong signal in the nucleus of DNC plated cells. At 48 hours there continues to be high
expression in the DNC cells, though the nuclear stain was not as clear as the 36 hour timepoint. NC cells
did not show nuclear staining. (B) DNMT3A nuclear localization is slightly affected by inhibitors of
transcription (actinomycin D) and translation (cyclohexamide) on NC, but downregulation on DNC
strongly depends on both functions (n=4). SMC were plated for 4 hours as in Figure 1 and treated with
cyclohexamide or actinomycin for the next 44 hours.
33
Figure 6 DNMT3A transfection does not alter subcellular localization
Primary huBSMCs were transfected with myc-DNMT3A and plated onto different matrices
following serum starvation (n=4). Cells were fixed at 48 hours and stained with a DNMT3A and
a myc-tag antibody. The endogenously expressed DNMT3A as well as the transfected myc-
DNMT3A showed nuclear staining on DNC and cytosolic staining on NC.
2.2.6 Matrix regulation of DNMT3A is cell density dependent
Previously, our lab reported that DNC can induce a high level of mitosis at 48 hours, in cells
initially culturing at a low cell density40. As BSMCs are proliferating at a higher rate on DNC, I
found that the DNMT3A nuclear localization is dependent on a low cell density (Figure 7 A).
Briefly, different cell densities ranging from 1 x 104 to 6.5 x 105 cells/mL were used to plate
BSMCs on DNC. Consistent with earlier experiments, cells plated at lower densities showed the
upregulation of nuclear DNMT3A expression. Conversely, cells cultured at higher densities
showed decreased nuclear expression of DNMT3A. To see whether mitosis is involved in this
response, I treated BMSCs plated on DNC with a mitotic inhibitor, nacodazole. This inhibitor
prevented the cells’ response to damaged matrix as the nuclear DNMT3A expression was
Ho
echst
DN
MT3
A
NC DNC
29.00 um
Myc-D
NM
T3A
34
attenuated (Figure 7 B). To further examine the role of mitosis we used two SMC mitogens,
epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), which are known to
induce SMC hyperproliferation. Interestingly, the effect of matrix seems to predominate as the
two SMC mitogens did not potentiate the nuclear expression of DNMT3A on DNC nor increase
the basal expression of DNMT3A on NC (Figure 7 C).
Figure 7: DNMT3A expression is regulated by cell-density, mitosis but not mitogenic growth
factors.
SMC were plated as described in Figure 1. (A) Cell density affects localization of DNMT3A to the
nucleus (n=3). (B) Nuclear expression of DNMT3A is decreased by the mitotic inhibitor nocodazole in
cells (n=4). (C) EGF (50 μg/mL) and FGF (10 μg/mL) fail to alter nuclear localization from patterns
established on NC or DNC (n=4).
35
2.2.7 Signaling pathways regulate Dnmt3a localization on damaged matrix
“Previously, we found that proliferation of SMC in response to obstructive stimuli including
damaged matrix is associated with several signaling pathways, including MEK/ERK 39 and
JAK2/STAT3 42.We also noted that JAK2 inhibitors uncoupled epigenetic modulation of
differentiation, decreasing DNMT3A localization and proliferation, but not -SMA expression
42” (directly quoted from 137) We first examined how inhibition of the MEK/ERK pathway affect
the DNMT3A localization. PD98059 was used to treat BSMCs after the initial plating onto on
different matrices (at 6 hours) and the inhibition of the MEK/ERK pathway lead to decreased
DNMT3A expression on both DNC as well as NC (Figure 8 A). The downregulation of SMC
differentiation genes on DNC was prevented. MEK signaling seems to affect the global
expression of DNMT3A.
Integrins, including 3 are important in mediating SMC’s response to changes in the
extracellular matrix/ phenotypic switching. We investigated the role of 3 signaling in this
response by blocking the integrin with an F11 antibody. At 48 hours, the F11 antibody
completely attenuated DNMT3A nuclear localization in BSMC on denatured collagen (Figure 8
B).
2.2.8 Inhibition of nuclear export did not alter DNMT3A localization on NC
and DNC
Since DNMT3A has a nuclear localization signal (NLS)139, the observed change in subcellular
localization could be due to either DNMT3A getting exported out of the nucleus on NC or
getting imported into the nucleus on DNC. We treated primary huBSMCs with a nuclear export
inhibitor Leptomycin B (20uM, Cell Signaling) as the cells were plated onto NC or DNC. The
cells were fixed at 24 or 48 hours with 4% PFA and stained with a DNMT3A primary antibody.
The treatment of Leptomycin B did not significantly alter the subcellular location of DNMT3A
in cells cultured on NC or DNC. At both 24 and 48 hours (Figure 9), the DNMT3A is localized
in the nucleus on DNC and in the cytosol on NC, with or without Leptomycin B treatment. This
suggests that DNMT3A is transported into the nucleus on DNC.
36
Figure 8: DNMT3A expression is inhibited by ERK and F11 inhibitors on DNC.
The ERK integrin pathway participates in matrix induction of DNMT3A (n=4). The pathway inhibitor of
ERK (40 μM PD985059) affects nuclear expression of DNMT3A, and prevents the loss of SMA and
myosin expression on DNC as well as on NC (A, B). (C) DNC induction of DNMT3A nuclear
localization is dependent upon integrin signaling (n=4). The blocking antibody F11, which prevents β3
integrin signaling, attenuated DNMT3A nuclear expression.
37
Figure 9 : Nuclear export inhibitor did not alter DNMT3A localization on NC
Primary human BSMCs were serum starved and plated onto different matrices with or without
the treatment of Leptomycin B (20uM)b. Cells were fixed at 24 hours and 48 hours and stained
with a DNMT3A antibody. The cytoplasmic DNMT3A expression on NC was not altered by the
nuclear inhibitor treatment.
38
2.2.9 Matrix induces significant changes in DNA methylation
“Damaged matrix is a persistent stimulus to bladder smooth muscle cells caused by bladder
obstruction in vivo. In order to examine DNA methylation events associated with a damaged
collagen matrix, we took a genome-wide approach using the Illumina 450 K methylation array to
probe bisulfite-converted DNA from [primary] human bladder smooth muscle cells” (directly
quoted from 137) First, I confirmed that the human BSMC showed a consistent pattern of
DNMT3A subcellular expressions as rat BSMC on different matrices (Figure 10 A). At 48 hours,
however, I did not observe an increase of DNMT3A mRNA expression.
“The DNA methylation array data was analyzed first by comparing methylation between the
groups from the two substrates using pre-filtering for a significant Welch’s t-test, and secondary
correction for multiple testing by Benjamini-Hochberg. The overall data distribution showed
minimal changes and a similar amount of hyper and hypo-methylation, with only a small number
of sites showing significant changes after correction for multiple testing (Figure 10 B).” (directly
quoted from 137) The DNC induced several significant and discrete methylation changes in
human BSMC after 48 hours of culturing (Table 4).
An a priori analysis was then performed for CpG sites that are related to SMC differentiation.
Higher levels of methylation in human BSMCs were observed on DNC compared to NC after the
correction for multiple testing (Figure 11 A). 14 CpG sites from 12 SMC related genes were
significantly hypermethylated (Figure 11 B). It is worth noting that although the methylation
changes are relatively small in degree compared to those observed in cancer studies, these
changes were induced by an otherwise benign disease model and after only 48 hours, and thus
may provide us with an initial epigenetic signature of this benign disease.
39
Figure 10: Damaged matrix induces DNMT3A nuclear expression in human bladder SMC and
changes in methylation status in CpG sites of the Illumina 450K methylation array.
(A) Human bladder smooth muscle cells were plated on native (NC) or denatured collagen (DNC) at low
density (4×104 cells/mL) for 6 hours in EMEM with 6% FCS, then media was changed to 2% FCS in
EMEM. Nuclear expression of DNMT3A is increased in SMC cultured on DNC (n=4). By
immunofluorescent staining, levels of DNMT3A and smooth muscle myosin heavy chain (MHC, smooth
muscle-specific form) were examined by spinning disk microscopy using Volocity software, then
analysed with Image J. DNMT3A and 3B were both examined by QPCR. While DNMT3A levels were
not significantly increased by mRNA expression, protein expression of DNMT3A and DNMT3B levels
were increased *, p<0.05 (n=4). (B) Illumina 450 K CpG methylation array of human SMC plated onto
NC and DNC show several significant changes at discrete hypomethylated and hypermethylated CpG
sites on DNC compared to NC. Red diamonds indicate significantly altered CpG methylation (adjusted
p<0.05, by Benjamini-Hochberg).
40
Table 4 Differentially methylated CpG sites
(T-test <0.01, adjusted p value < 0.05, Benjamini-Hochberg) revealed after analysis of specific
regions or all sites of the epigenome
41
Figure 11: A priori test of CpG sites in SMC specific genes reveals specific changes in DNA
methylation.
(A) Volcano plot of hypomethylated and hypermethylated CpG sites reveals a clear trend toward
hypermethylation of sites in cells plated on DNC. 14 CpG sites have statistically significant increase in
methylation. (B) Beta values (degree of methylation) in 14 CpG sites near 12 genes differed between cells
cultured on NC and DNC. Differences between cells on native collagen and denatured collagen were
significantly altered in all sites (adjusted p value <0.05).
42
3 Chapter 3: Gene expression is persistently altered in
irreversible bladder obstruction
3.1 Methods
3.1.1 Bladder obstruction and release
“The experiment protocol used was approved by the institutional animal care committee in
accordance with policies established in the Canadian Council on Animal Care Guide to the Care
and Use of Experimental Animals.” (directly quoted from 28) In a previous in vivo study (study
1), partial bladder obstructions were created in female Sprague-Dawley rats. A small incision
was made to expose the proximal urethra. A 0.9mm diameter metal bar was placed alongside the
urethra and a suture was used to tie both the rod and the urethra to prevent complete obstruction.
Subsequently, the bar was removed leaving a constriction of a standardized size around the
bladder neck.
At the end of the 6 week obstruction period, a group of obstructed rats were sacrificed for
bladder harvest (obstruction only control, OBX) while the other obstructed animals underwent
deobstruction surgeries and were allowed to recover for another 6 weeks (obstruction followed
by release, BRV). At the end of 12 weeks, both sham operated (12sham) and BRV rats were
sacrificed and their bladders were harvested. After opening the abdomen, the residual urine
volume was measured. The bladder weight as well as the body weight was also recorded.
It was discovered at a later point that the RNA from some bladders had been degraded and thus
not fit for downstream experiments such as the RT2 profiler array PCR array (Qiagen). The RNA
degradation was likely caused by the repeated freeze-and-thaw cycle prior to RNA isolation.
After consulting with Qiagen’s technical support, our lab decided to add more samples to the
original sample collection. The additional samples were selected from bladder obstructions that I
had created (study 2). Briefly, female Sprague-Dawley rats underwent sham or obstruction
surgery as described above and were sacrificed at the end of 2 weeks (short term obstruction) or
43
6 weeks (long term obstruction). Only 6 week obstruction (OBX) or sham samples (6sham) were
added to study 1.
Due to the low number of biological replicates from each group and the variability amongst the
samples, we decided to validate the expression data of persistently dysregulated genes using
additional study 1 samples. Five to six samples were selected from each treatment group. (See
table 5 for a full list of bladder samples)
Table 5 Sample groups
Group Treatment Study Application
6sham 6 week sham control 2 PCR array
12sham 12 week sham control 1 PCR array and
RT-PCR validation
OBX 6 week obstruction only 1 and 2 PCR array and
RT-PCR validation
OBR 6 week OBX + 6 week release 2 PCR array and
RT-PCR validation
3.1.2 RNA and DNA isolation
In study 1, harvested bladders were cut into domes and bases. Cut samples were immediately
snap frozen using a dry ice-ethanol bath and stored at minus 80°C. To isolate RNA, bladder
dome was homogenized and processed using a Bullet Blender® and TRIzol (Life Technologies)
according to protocols. The RNA is dissolved in DNase/RNase free water and quantified.
In study 2, harvested bladders were immediately incubated in RNAlater (Life Technologies) on
ice and then at 4°C overnight (as instructed by manufacturer’s protocol). The samples were
stored in -80°C with the RNAlater solution. To isolate RNA, half of the bladder dome was
thawed on day one, dried and re-frozen in -80°C overnight. On day two, the sample was first
crushed and grinded by a chilled mortar and pestle (-80°C overnight), then further homogenized
in the TRIzol Reagent by using the Qiagen Tissuelyzer. RNA was then extracted as per
manufacturer’s protocol. The integrity of the RNA was checked using the Bioanalyzer (Agilent
2100, processed by The Center for Applied Genomics at the Hospital for Sick Children). Only
samples with RNA Integrity Number greater than 8 were used.
44
RNA extraction for the PCR validation sample set was conducted in the same manner as samples
in study 2.
3.1.3 Custom PCR Array (with additional samples)
Our lab had curated 88 genes (Table 6) related to bladder injury and ordered custom made PCR
array plates from Qiagen (CAPR11774) to examine their mRNA expressions across different
treatment groups. Each 96-well plate contains primers for 88 selected genes, housekeeping
genes and built in quality controls.
For each bladder sample, 800ng of RNA was used for cDNA synthesis with the RT2 First Strand
Kit (Qiagen) according to the manufacturer’s protocol. The cDNA was then mixed with the
appropriate amount of RT² SYBR Green ROX qPCR Mastermix (Qiagen) and dispensed into a
PCR plate. Real-time PCR was performed with suggested cycling conditions. The expression
data was analyzed using the delta-delta cT method as per protocol. Different treatment groups
were normalized to both the 6 week sham (6sham, shams from my sample set) as well as the 12
week sham (12sham, shams from the original sample set).
3.1.4 Validation of expression changes
For the validation real time-PCR (RT-PCR) experiment, cDNA was generated as described
above and around 5 ng of cDNA was used per reaction. All samples were tested in triplicates
and real-time PCR was performed with suggested cycling conditions. The expression data was
analyzed using the delta-delta cT method as per protocol.
45
Table 6 Genes with CpG islands, curated from rat bladder obstruction literature
46
3.1.5 Pyrosequencing
DNA was extracted using the phenol-chloroform extraction (Sigma Aldrich) according to protocol. DNA
was quantified with Nanodrop, checked for integrity on electrophoresis gels and submitted to Dr.
Weksberg’s lab for pyrosequencing. 20 CpG sites within the CpG island upstream of the KCNB2 promoter
47
region were examined140. Up to 1 ug of DNA was bisulfite converted using the EpiTect Plus DNA Bisulfite
Kit (Qiagen) as per manufacturer’s protocol. The degree of DNA methylation at each CpG site was
analyzed using the PryoMark Q24 (Method 006). A total of 7 out of the 20 CpG sites showed readouts that
passed the built-in quality check and were subsequently analyzed.
3.2 Results
3.2.1 Release of 6 week bladder obstruction does not completely reverse the
bladder/body weight ratio or functional parameters
Consistent with previous studies, obstruction induced a significant increase in bladder weight
(normalized to body weight) at the end of 6 weeks post obstruction (Figure 12). The level of
bladder hypertrophy was decreased by de-obstruction followed by 6 weeks of recovery but still
remained higher than the sham operated group. It is worth noting that even though the
obstruction-release group has a much lower bladder weight compared to the obstruction only
control, there is still roughly a 2 fold change in bladder weight compared to the sham, which is
enormous for a rat and therefore considered persistent pathology.
48
Figure 12 Bladder obstruction significantly upregulates bladder mass
Partial bladder outlet obstructions were created in female Sprague-Dawley rats for 6 weeks, with
(OBR) or without (OBX) 6 weeks of de-obstruction. At the time of sacrifice, obstruction only
bladder exhibited massive increase in bladder weight (per body weight). De-obstruction for
additional 6 weeks allowed partial recovery of bladder hypertrophy, but OBR bladder remains
bigger than sham controls.
3.2.2 6 week bladder obstruction leads to dysregulation of genes that are
persistent even after release
The previous in vivo study (study 1, operated by Annette Schroder) included a long-term bladder
obstruction group (6 weeks), a long-term bladder obstruction followed by release group (6
weeks plus 6 weeks release) and a sham operated control group (12 weeks). The mRNA
expressions of 88 SMC differentiation related genes were compared using the RT2 custom PCR
49
Array plates (Qiagen). Due to RNA quality issues, 6 week obstructed and sham operated
bladder samples (operated by me) were added to the sample set. Only samples that passed the
array’s built-in quality checks were included in the analysis and the data was quantified using
the delta-delta cT method. KCNB2 and DNMT3A is significantly altered in both of the
obstruction only group as well as the obstruction followed by release group when normalized
against the 12 week sham group (Figure 13 A) Similarly, HIF1 and DNMT3B is
significantly altered in both treatment groups compared to the 6 week sham group.
Due to the low number of biological replicates from each group and the variability amongst the
samples, we decided to validate the expressions of the four persistently dysregulated genes using
additional samples from the in vivo study 1. Five to six samples were selected from each
treatment group. The RNA from each sample was extracted and cDNA synthesized as described
above. Compared to the sham, KCNB2 is significantly downregulated by bladder obstruction
with or without release (Figure 13 B). However, the comparison of HIF1 mRNA expressions
between different groups did not show statistical significance.
50
Figure 13: Long term obstruction causes persistently dysregulated gene expression that is
not reversed by de-obstruction
A. Relative mRNA expression comparisons of genes across treatment groups as detected by the
custom PCR array. KCNB2, HIF1, DNMT3A and DNMT3B were significantly altered during
obstruction and after de-obstruction. The two control groups, however, show opposite trends of
expression for all four genes. B. The mRNA expression profile of KCNB2 and HIF1 was
verified by traditional RT-PCR using additional samples. Only KCNB2 expression changes
remained statistically significant (n=5).
51
3.2.3 Pyrosequencing
The degrees of DNA methylation at 20 CpG sites in the island upstream of the rat KCNB2
promoter were analyzed using PyroMark Q24. (Figure 14 A) Readout signals at the first 7 CpG
sites passed the quality check for most biological samples (Table 7) and the average percent
methylation for these 7 sites was calculated. (Figure 14 B) The degrees of DNA methylation
were highly variable within the obstruction only group (OBX). We did not observe any
significant difference in methylation states at the 7 sites. Analysis of the per cent methylation at
each individual CpG site showed statistically significant difference between the OBX group and
the 12sham group at position 3 (p = 0.036). The methylation difference between the BRV and the
12 sham at this position showed a decreasing trend (p=0.058)
52
Figure 14: DNA methylation states of 7 CpG sites upstream of KCNB2 were not changed
A. The CpG island upstream of KCNB2 gene was selected for pyrosequencing analysis
(Genomic size 832 bp, with 103 CpG sites). B. 7 CpG sites (out of 20) passed quality checks for
most samples, there was no significant difference in the average methylation states between any
groups. The degrees of DNA methylation were highly variable within the obstruction only group
(OBX). We did not observe any significant difference in methylation states at the 7 sites.
Analysis of the per cent methylation at each individual CpG site showed statistically significant
difference between the OBX group and the 12sham group at position 3 (p = 0.036). The
methylation difference between the BRV and the 12 sham at this position showed a decreasing
trend (p=0.058)
53
Table 7. Percent methylation at each CpG site
Pos.1 Pos. 2 Pos. 3 Pos. 4 Pos. 5 Pos. 6 Pos. 7
12sham 1 3.05 0.68 3.05 3.15 1.87 4.73 1.37
12sham 4 1.83 1.81 0.93 0.65 1.31 6.23 1.23
12sham 7 3.3 1.74 1.06 1.22 1.23 2.53 4.27
OBX 3 0.95 2.02 0 0 0 0.63 0
OBX 4 4.48 0 0.91 0.39 0.66 6.85 0
OBX 10 0.49 0 0.65 15.07 17.93 1.38 0.9
OBX 5 1.95 1.29 0.79 0.59 1.5 1.76 0.76
OBX 6 1.53 1.42 1.57 1.28 1.55 1.91 0.74
BRV 1 3.14 0.69 0.59 0.6 4.83 2.12 2.51
BRV 3 0.93 0.45 1.13 1.18 2.97 3.77 0.53
BRV 5 3.01 0.85 0.51 1.08 3.91 1.99 0.65
BRV 12 2.52 1 1.14 0.65 2.51 2.04 1.54
54
4 Chapter 4: Discussion
4.1 Matrix and SMC biology
4.1.1 Control of DNMT3A localization
The DNMTs have N-terminus nuclear localization signals (NLS.) As with other NLS containing
proteins, the acetylation of lysines in the NLS peptide sequence may alter their translocation by
nuclear import proteins and thus their cytoplasmic retention141-144. Several studies have reported
the cytosolic retention of DNMTs145,146 (reviewed by137). The DNMT3A is imported by alpha-
importins, which also have an NLS sequence that can be regulated by acetylation147-149 (reviewed
by137). Under normal physiological conditions, the microenvironment should support
differentiated cells with mostly cytoplasmic DNMT3A since functional adult somatic cells
should be relatively quiescent and terminally differentiated. Based on our present observations, it
is worth noting that previous in vitro immunocytochemistry and western blot studies have used
tissue culture as a substrate, which induces cell behaviors similar to those plated on denatured
collagen. Cells grown on tissue culture plastics may have enhanced nuclear localization of
DNMTs and may not show the accurate and relevant expression of cytosolic DNMT137.
On the other hand, most of the DNMT subcellular localization research has used cancer cell lines
and embryonic stem cells rather than primary cells, different cell types/lines may have unique
matrix adherence effects. The change in DNMT localization in response to a pathological
stimulus was also observed in urothelial cell lines150. In a bacterial infection model, cytosolic
DNMT1 was observed in urothelial cell lines on tissue culture plastic. The urothelial cells
showed nuclear upregulation of DNMT1 in response to inoculation with pathogenic bacteria (but
not with the non-pathogenic bacteria). The change in DNMT3A localization on DNC (vs. NC)
suggests that the DNA methylation machinery is altered by changes in the microenvironment
because more of the DNA modifying enzyme is present in the nucleus. Proteosomal degradation
of nuclear proteins such as DNMTs is regulated post-transcriptionally, which includes
phosphorylation by GSK3 and ubiquitination by the proteosomal pathway151-154 (reviewed
55
by137). Degradation would decrease the total amount of DNMTs available for nuclear
localizations and DNMTs should be retained and eventually degraded in the cytoplasm.
Opposite DNMT3A subcellular locations on the two matrices could be mediated in two ways: 1)
active nuclear transport of DNMT3A on DNC only; 2) the default nuclear transport (based on the
NLS signal) on both collagen substrates, followed by the active nuclear export of DNMT3A on
NC. “ Chromosomal region maintenance/exportin 1 (CRM1) exports HDAC1 to the kinesin
motors of the cytoplasm, blocking motor activity155. This opens the possibility that HDACs or
other molecules associate with and shuttle DNMT to different cellular compartments.” (directly
quoted from 137) Leptomycin B is a potent and specific inhibitor of CRM1 and is generally used
for nuclear export studies130,156. Treatment of Leptomycin B did not alter the DNMT3A
localization pattern on different matrices. The absence of nuclear DNMT3A staining on normal
collagen indicates that the differential subcellular translocation is not mediated by default nuclear
import followed by nuclear export on NC, but rather by the active nuclear import on DNC.
4.1.2 MMP remodeling during fibroproliferative disease
“In vivo, fibroproliferative stimuli are inextricably linked in hollow organs (e.g. bladder, heart,
and vasculature), with mechanical strain including the expression and activation of matrix
metalloproteinases (MMPs), particularly the gelatinases (MMP2 and MMP9), which can
profoundly alter the matrix microenvironment 39,155,157” (directly quoted from 137) Due to the
slow turnover rate of its components, the ECM can exert long term influence on SMC phenotype
modulation158. Mechanical strain and excessive pressure can compress the microvasculature in
hollow organs and lead to tissue hypoxia159,160. Hypoxia activates MMP7 expression that alters
the ECM components39. Interestingly, as one of the inciting stimuli during bladder obstruction,
hypoxia only potentiated the matrix’ modulation of DNMT3A localization and hypoxia alone did
not induce nuclear localization. Rapamycin, given immediately after cell adherence, was able to
prevent the de-differentiation in our previous study39.
On denatured collagen, rapamycin could not reverse the loss of SMC differentiation marker and
cell hypertrophy without DAC treatment. Together the two treatments reversed the SMC
phenotypic switching on DNC. This demonstrates that even though rapamycin can help prevent
56
the pathological changes during disease progression, it alone is not able to reverse the phenotypic
switching process once SMC injury develops to a certain degree.
“On DNC alone, the localization of DNMT3A is dependent on cell density. We speculate that
this might relate to either the level of mitosis in the cells, the degree of paracrine/ autocrine
signaling, or cell-cell contacts. At higher densities, cells may decrease mitotic activity or increase
their autocrine signaling and cell-cell contacts.” (directly quoted from 137) Pathways related to
the level of mitosis and cell cycle regulation seem to predominantly regulate the expression level
and intracellular trafficking of DNMT3A. Cell cycle arrest, induced by Nacodazole, prevented
the nuclear localization of DNMT3A on DNC while the addition of FCS prior to plating induced
DNMT3A localization on NC. Therefore, DNA methylation change depends on the level of
mitosis. Hyperproliferation, a pathological phenotype of the BSMC during bladder obstruction,
may be mediated by changes in DNA methylation. The level of mitosis can affect the DNA
methylation changes and vice versa but the exact mechanism(s) is unknown. Interestingly, two
well-known SMC mitogens (EGF and bFGF) however, did not alter the expression or trafficking
of DNMT3A on different matrices even though they have been shown to induce SMC
phenotypic changes161-164. In this experiment, cells were plated at a low density prior to the
mitogen treatment, the response to EGF and bFGF may be prevented due to the existing, DNC
induced hyperproliferation. However, NC renders BSMCs quiescent therefore the lack of
response to mitogens is not clear. The EGF and bFGF treatment to BSMCs plated at a high cell
density will be an important experiment to determine whether mitosis can induce DNMT3A
nuclear localization.
Alternatively, the intracellular trafficking of DNMT3A may relate to cytoskeleton arrangement.
As a cell cycle inhibitor, Nocodazole prevents cell division by interfering with microtubule
formation165 during cytokinesis. The decreased nuclear DNMT3A expression may result from
the impaired intracellular trafficking as the microtubule assembly is inhibited. A cytoskeletal
protein extraction of BSMCs plated on different matrices will determine whether DNMT3A
physically associate with the cytoskeleton.
57
4.1.3 Epigenetic mediation of disease progression
The significance of epigenetic mediation in SMC disease is beginning to be appreciated with
several studies published but relatively few papers examined the role of DNA methylation during
this process. As mentioned, PDGF-induced mitogenic activity in SMC is dependent upon
epigenetic mechanisms128. In an in vitro PCR array study, SMC differentiation was coupled with
a trend toward DNMT downregulation166. The study by Hodges showed the regulation of
collagen type I and III gene expression in neurogenic bladders was at least partially mediated by
histone modification as well as by DNA methylation125. In obstructive uropathy, the BSMCs
have persistent influence on ECM deposition and we have shown in addition that the ECM has
long lasting regulatory roles in SMC phenotypic modulation. “In the present study, DNC with
and without hypoxia increases DNMT3A localization and decreases SMA expression. It will be
important to understand how the context of different inciting stimuli alters the regulation of
DNMT3A along with its histone and transcriptional co-factors. DNA methylation of some sites
may be beneficial, while methylation of others is detrimental in these contexts. Nonetheless, the
crucial role of matrix in all of the contexts examined here suggests that matrix is a crucial
component for upregulation of the DNA methylation machinery in non-malignant cells.”
(directly quoted from 137)
4.1.4 Matrix alters DNA methylation in BSMC
“Matrix can rapidly alter methylation of distinct CpG sites, as our array experiment duration was
only two days. Despite known limitations with CpG array technology 167, the changes in β
values at various sites after a short exposure time to matrix suggests that the extracellular matrix
environment may, in part, exert its effects on regulation of gene expression through alterations in
DNA methylation168. The number of differentially methylated CpG sites is within the range of
changes seen in other MethylArray comparisons, such as dilated cardiomyopathy and end-stage
heart disease134,169. In contrast, in one study by Sandoval et al, 2011 170, the comparison of colon
cancer cells with 2 different normal colonic mucosae yielded only 3–6% of sites with differential
DNA methylation. While the latter study compared cancer cells and normal tissue, our work
examined differences in methylation in one primary cell line plated in two different
environmental conditions over a relatively short period of time. In contrast to cancer cell lines or
58
tumor tissues, the cardiomyopathy studies revealed only very discrete changes, with the majority
of sites failing to show any dysregulation using standard statistical methodologies. The heart
studies and our own utilize non-cancer cells or tissues, which, unlike cancer cells, still retain
many of the epigenetic controls for cell differentiation. In this context then, it is actually quite
striking to observe discrete alterations at 14 SMC differentiation related sites over the course of
only 48 hours.” (directly quoted from 137) The degree of changes were expected to be small
because our lab uses primary cells in a disease model that is otherwise benign compared to
cancer cells.
4.1.5 Future directions
“By examining how SMC in vitro respond to matrix to cause long-term changes, our goal is to
identify therapeutic targets and biomarkers for intractable disease through an examination of
DNA methylation patterns.” (directly quoted from 137) How matrix regulates DNMT expression,
subcellular localization and activity is crucial for the understanding of the epigenetic instigators
underpinning fibroproliferative diseases.
To elucidate the epigenetic signaling effectors in response to matrix stimulation, we used
different signaling inhibitors in this study. The integrin v3 mediates cellular response to
changes in the microenvironment and its inhibition by F11 antibody abrogated nuclear
DNMT3A expression on DNC. RGD peptides are exposed during ECM degradation 164 and are
shown to activate signaling pathways (e.g. AKT and ERK) via v3 induction171-173. AKT
signaling is involved in cardiomyocytes hypertrophy in vivo 174 and can lead to epigenetic
silencing of genes via the polycomb-repressive complex (PRC) mediated histone
modification175-177. Preliminary data (Appendix II, Figure 22) shows that AKT signaling is
upregulated at early timepoint (3 hour) on DNC, but not at later timepoint (48 hour). At 3 hour
post plating, the mTOR pathway is also activated. Elk is downstream of mTOR signaling, it is
also a transcriptional co-activator of SRF 47 that induces SMC de-differentiation. Therefore,
future studies should examine if AKT signaling affects SMC’s epigenetic response to changes in
the microenvironment.
59
An alternative approach to identify candidate pathways that mediate DNC induced methylation
changes was to contrast physical interacting proteins of DNMT3A on NC vs. DNC. BSMCs
were transfected with a myc-tagged DNMT3A bait and the binding complexes isolated from
immunoprecipretation (IP) were analyzed by mass spectrometry. Unfortunately after
optimization of the matrix disease model with transfected bSMC (See Appendix I for detail), it
was clear that IP followed by mass spectrometry analysis demanded unfeasibly large quantities
of collagen substrates and transfected cells. Future experiments can be planned to select for cells
with stable expression of the plasmid using a selection marker or stably express the plasmid
using lentiviral transduction.
4.2 Irreversible bladder obstruction
4.2.1 Irreversible bladder obstruction and persistent gene dysregulation
Prolonged duration of bladder obstruction in patients can often lead to incomplete anatomical
and functional recovery of the bladder even after relief of obstruction. This partial irreversibility
is also observed in prolonged bladder obstruction in animal studies and the underlying
mechanism remains unclear. In the early stages of obstruction the bladder has increased muscle
mass, ECM deposition, increased wall thickness and decreased lumen size. Prolonged
obstruction leads to a decompensatory phase in which the bladder assumes a distended shape
with increased lumen volume, loss of contractility and compliance7. In a rat bladder obstruction
study, bladder weight and wall thickness after 3 weeks of obstruction were significantly higher
than the sham control group178. In the animals that underwent 3 weeks of obstruction followed by
release of obstruction for an additional 3 weeks, the bladder weights had returned to levels
similar to the sham group at the time of sacrifice. In a study by Malmqvist et al., rats which had
undergone bladder obstruction for a longer period (7 weeks), followed by the removal of
obstruction and additional 7 weeks of recovery, continued to have higher bladder weights and
60
impaired bladder functions compared to control5. Therefore by varying the duration of bladder
obstruction in animals we can create irreversible and reversible bladder obstructions.
Bladder obstructive disease in patients is most often diagnosed after a significant period of time
has lapsed since the onset of the obstruction. In this study we created long term bladder
obstructions, with or without release and recovery, to examine whether SMC differentiation
related genes are persistently dysregulated even after the ablation of anatomical obstruction.
Quantification of the custom array data using the delta-delta cT methods showed 4 persistently
dysregulated genes (KCNB2, HIF1, DNMT3A and DNMT3B), only KCNB2 expression
changes remained statistically significant in the subsequent RT-PCR validation of KCNB2 and
HIF1. Voltage gated potassium channels have important roles regulating SMC contractions
through the modulation of membrane potential and voltage gated calcium channel activities164.
Even though its transcriptional upregulation did not remain statistically significant, the hypoxia-
inducible factor (HIF1) is a key mediator of hypoxia induced injury during bladder
obstruction179,180. Instead of persistent transcriptional upregulation, the predominant regulation of
HIF1activity may occur at the protein expression level in an oxygen content dependent
manner181.
A study that examined the gene expression changes before and 10 days after the relief of bladder
obstruction showed many more commonly altered genes compared to the control group182. (See
Table 7 in Appendix II) Given the short duration of recovery, however, the majority of these
genes may not be persistently dysregulated at a later point. Furthermore, the expression of
hypermethylated genes (from the damaged matrix array, Section 2.2.9) can be tested in all
groups to understand how the gene expression changes with the obstruction and the de-
obstruction process.
Our initial pyrosequencing results showed no significant change in the average of percent
methylation in 7 CpG sites. The degree of methylation is significantly higher in the obstruction
only group compared to the sham control (Section 3.2.3). Currently, no transcription factor
binding data for rat is available on the USCS genome browser. Examination of the human
KCNB2 gene, however, showed that the gene also has a CpG island (similar size to the one
found in rat) upstream of its promoter140. ChIP-seq data shows Human enhancer of zeste 2
(EZH2) binding overlapping with the CpG island and the observed hypomethylation could
61
decrease KCNB2 expression by enhancing EZH2 binding (EZH2 catalyzes repressive histone
modifications).
There are several reasons to explain why only a few irreversible gene expression changes were
observed. Genes examined in the custom array were curated largely from the bladder obstruction
literature only, with no particular insight into whether they were related to the reversal or
persistence of pathology. Secondly, our data has a low number of biological replicates and our
obstruction model usually has a high degree of variability (discussed below). Lastly, many genes
were excluded from the array data set due to their high cTs across all treatment groups. Some
gene expression changes may not be detected due to the quality of the array primers. BDNF, for
example, had very high cTs and therefore was omitted from the data set according to
manufacturer’s protocol. However, our lab has observed the persistent upregulation of this gene
during obstruction in another study.
4.2.2 Variability in animal models of bladder obstruction
Many different animal models of bladder obstructions have been developed to study the
molecular basis for the development of bladder dysfunction. Spinal cord transection is a common
model used to study neurological bladder obstructions as bladder hyperreflexia immediately
follows the injury182. The obstructive uropathy develops easily and consistently, and is relevant
to patients with spinal cord injury182. Another common model used is the creation of anatomical
partial bladder obstruction by standardized constriction around the bladder neck. The partial
outlet obstruction model replicates many architectural, physiological and the underlying
molecular bladder changes occurring in human patients. However, biological replicates within
the obstructed group can have varying levels of obstruction in our study as well as in other
studies. One study, for example, separated obstruction samples based on the degree of severity
before functional and histological analysis6. The “mild obstruction” group (vs. the “severe
obstruction” group) had functional parameters similar to the controls, suggesting some
obstructions were not successfully achieved. Bladder obstruction by spinal cord transection may
be a more consistent model in this regard. Moreover, outlet obstruction studies are generally
performed in female rodents. Females constantly experience estrogen fluctuations due to their
reproductive cycles and estrogens induce molecular changes, sometimes via epigenetic
62
mechanisms, in different cell types including cardiomyocytes and SMC183-186. Therefore, future
examinations of epigenetic changes caused by bladder obstruction should be conducted in male
animals despite even though the urethra is more difficult to expose as it situates behind the
prostate in males.
4.2.3 Future directions
Our study showed that DNA methylation signatures at 7 CpG sites upstream of KCNB2
promoter region did not show significant changes in methylation. More sequencing should be
conducted to determine if the observed gene dysregulation is mediated by the changes in DNA
methylation. The rat KCNB2 gene has 103 CpG sites in the CpG island that is upstream of the
promoter region140. In our initial probing, we examined 20 sites but only 7 sites passed the array
quality check and were analyzed. The DNMT3A and DNMT3B expression changes detected in
the PCR array should be validated and the DNA methylation profile should be examined if these
two genes are persistently dysregulated. Furthermore, the other two mechanisms of epigenetic
modifications should also be examined; the expression of microRNA 29 (miR-29) was altered
during bladder obstruction and its target genes related to ECM remodeling and detrusor
contractility were altered in a miR-29 dependent fashion126.
In syngeneic animal models of bladder obstruction, the level of recovery following de-
obstruction can vary. The ultimate goal is to uncover predictors for the degree of recovery in a
clinical setting or to identify potential therapeutic targets to reverse the seemingly irreversible
pathology. The mTOR is a key signaling pathway mediating pathological progressions of many
cardiovascular and SMC related diseases 24,187 and our lab has shown that treatment of rapamycin
during the development of bladder obstruction can signficiantly preserve bladder ECM integrity
and muscle functions28. However, the drug treatment was given during the obstruction in animals
whereas in patients the treatment usually starts after the pathology has taken its course. The
treatment of rapamycin after de-obstruction was not able to reverse physiological parameters
(data not shown). This observation is consistent with our in vitro finding as the treatment of
rapamycin alone after culturing on DNC was not able to rescue SMC phenotype. (Figure 1. B)
The combination of rapamycin and DAC, however, improved the SMC differentiation. Therefore,
it would be of interest to investigate how epigenetic intervention, with or without the inhibition
63
of mTOR pathway, mediates the progression as well as the recovery from bladder outlet
obstruction.
5 Table 8: Abbreviations
-SMA -smooth muscle actin
bFGF basic fibroblast growth factor
BPH benign prostate hyperplasia
BrdU bromodeoxyuridine
BSMC bladder smooth muscle cell
CRM1 Chromosomal region maintenance/exportin 1
DAC 5-Aza-2'-deoxycytidine
DNC denatured/damaged collagen
DNMT DNA methyltransferase
ECM extracellular matrix
EGF epidermal growth factor
ERK extracellular signal-regulated kinase
EZH2 Human enhancer of zeste 2
FCS fetal calf serum
HDAC histone deacetylase
huBSMC human BSMC
HIF1 hypoxia-inducible factor
KCNB2 potassium voltage gated channel subfamily B member 2
LB luria broth
MMP matrix metalloproteinase
mTOR mammalian target of rapamycin
NC native/normal collagen
NLS nuclear localization signal
PDGF platelet-derived growth factor
PFA paraformaldehyde
PRC polycomb repressive complex
PUV post urethral valve
SERCA sarcoplasmic reticulum Ca2+-ATPase
SMC smooth muscle cell
TSA trichostatin A
VSMC vascular smooth muscle cell
64
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Appendices
Appendix I: Human BSMC Transfection
Rationale
Previously, we observed that aberrant matrix microenvironment alone can incite a stable
phenotype alteration in BSMC, which is not completely reversed upon the return to normal
matrix. To elucidate candidate pathways that mediate DNC induced methylation changes,
DNMT3A-interacting proteins in cells plated on NC vs. DNC should be curated and contrasted.
I attempted to transfect huBSMC with a myc-tagged DNMT3A bait and isolate as well as
identify the DNMT3A binding complexes by immuneprecipitation (IP) and mass spectrometry
(MS).
Material and Methods
HuBSMC transfection
The plasmid pcDNA3/Myc-DNMT3A was a gift from Arthur Riggs (Addgene plasmid # 35521)
and was amplified as well as verified as mentioned above.
To transfect cells using Lipofectamine 1000 (Life Technologies), huBSMCs at approximately
80% confluency were trypsinized and pelleted as described above.
To transfect huBSMCs with the Nucleofector II System (Lonza), cells at approximately 80%
confluency were trypsinized and pelleted as described above. To select the appropriate
Nucleofector Program on the machine, 5 different programs plus a no-program control were
tried. For each reaction, 2 x 106 cells were transfected with 2 ug of GFP plasmid (included in the
kit) according to the protocol. GFP expressions were visualized at 24, 48 and 72 hours and the
best program was selected. HuBSMCs were then transfected with 0.5 ug, 1.0 ug and 2.0 ug of
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the myc-DNMT3A plasmid, plated on chamber slides (BD Falcon) and cultured in Smooth
Muscle Cell Medium (ScienCell). Cells were fixed at 24 hour, 48 hour and 72 hour time points
and stained with an anti-myc-tag primary antibody (1:200, Abcam). Alternatively, at 48 hours
post transfection (with or without additional 24 hours of serum starvation), cells transfected with
myc-DNMT3A (myc-3A-BSMC) were trypsinized and plated onto native or denatured collagen
and fixed for immunostaining at 24 hours as well as at 48 hours.
Immunoprecipitation
HuBSMC cells were transfected with myc-3A plasmid and isolated at 48 hours post transfection.
6x106 or 1x 107 cells were used for an IP experiment. Protein lysates were isolated using a
Nuclear Extraction kit (Active Motif) and the IP was performed using myc-tag agarose beads
(Sigma) according to manufacturers’ protocols. The IP product was run on a western gel and a
myc-tag primary antibody (1:1000, Cell Signaling) was used.
Results
HuBSMC transfection using Lipofectamine was not efficient
HuBSMCs were transfected with myc-DNMT3A plasmids and fixed at 24 hours and at 48 hours
post transfection. There was no observable myc-DNMT3A expression at 24 nor at 48 hours
(Figure 15 A, B). Since the primary myc-tag antibody produced a high level of background, I
transfected huBSMC with a green fluorescent protein (GFP) plasmid for better visualization of
the plasmid expression. Transfected cells showed no significant amount of GFP at 24, 48 or 72
hours (Figure 16).
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Figure 15 Transfection using Lipofectamine
Primary hBSMCs were transfected with myc-DNMT3A using Lipofectamine 1000 (Life
Technologies) according to manufacturer’s protocol. Cells were fixed at 24 and 48 hours and
stained with a myc-tag primary antibody. No detectable amount of plasmid expressed proteins
were detected.
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Figure 16 GFP transfection using Lipofectamine
Primary hBSMCs were transfected with a GFP plasmid using Lipofectamine 1000 (Life
Technologies) according to manufacturer’s protocol. Cells were visualized at 24, 48 hours and
72 hour. The level of GFP fluorescence was not detected.
Transfection using the Nucleofector II system lead to higher transfection
efficiencies
First, cells were transfected with 2 ug of GFP plasmid and visualized under a microscope at 24
hours. All 5 programs produced a great amount of GFP compared to the no program control
(Figure 17). The program with most GFP expression (#4, P-024) was chosen for all subsequent
transfections. Since myc-DNMT3A transfected huBSMCs would need to be re-plated onto
different matrices, GFP transfected cells were serum starved for 24 hours or passaged once.
Both the starved and the “P1” cells show decreased but still detectable amount of GFP (Figure
18 A, B).
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Subsequently, huBSMCs were transfected with different amount of myc-DNMT3A plasmid and
visualized at 24 hours, 48 hours and 72 hours post transfection using an anti-myc-tag primary
antibody. At 24 hours, 1.0 ug seems to be the optimal amount of plasmid per reaction (Figure 19
A) It appears that 2 ug of plasmid per reaction was toxic for the cells as suggested by the low
number of cells per field and 0.5 ug of plasmid per reaction had lower transfection efficiency.
The transfection efficiencies increased at 48 hours and decreased at 72 hours (Figure 19 B and
C). Therefore, the optimal transfection condition is to use 1.0 ug of myc-DNMT3A plasmid per
reaction (2 x 106 cells per reaction) and the highest efficiency is achieved at 48 hours.
Figure 17 GFP transfection with Nucleofector
Primary hBSMCs were transfected with a GFP plasmid using the Nucleofector II system (Amaxa)
according to manufacturer’s protocol. Five different transfection programs were tried. Cells were
visualized at 24 hours and intense GFP signals were observed. Program 4 yielded the highest
GFP expression.
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Figure 18: Sub-passage decreases plasmid expression
Primary huBSMCs that were transfected with a GFP plasmid were sub-cultured at 24 hours (A)
and 48 hours (B) for an additional day. The GFP expression was greatly reduced at both
timepoints.
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85
Figure 19 : Myc-DNMT3A plasmid expression is highest at 48 hours.
Primary hBSMCs were transfected with the myc-DNMT3A plasmid using the Nucleofector II
machine (Amaxa) according to manufacturer’s protocol (n=3). Three different amounts of
plasmids were tried. Cells were fixed at 24, 48 hours and 72 hours post transfection and
visualized with a myc-tag antibody. The highest tranfection efficiency occurred at 48 hours using
1 ug of myc-DNMT3A plasmid.
Serum starvation is required prior to plating onto matrices
The expression of transfected plasmid is generally influenced by the level of mitosis in the cells.
As we have shown previously, the subcellular localization of DNMT3A is dependent on the level
of mitosis (Figure 20), I asked if serum starvation before plating onto NC and DNC is required
to preserve our current model. At 48 hours post transfection, huBSMCs expressing the myc-
DNMT3A plasmid (myc-3A-BSMC) were either plated directly onto NC and DNC using 1%
FCS EMEM, or serum starved for 24 hours followed by plating onto different matrices. At the
end of 48 hours, cells on NC or DNC were fixed and stained with a DNMT3A primary antibody.
As shown above, previously starved myc-3A-BSMC had consistent DNMT3A localizations with
the non-transfected BSMCs, however, the cells plated directly onto different matrices without
starvation showed weak nuclear localization of DNMT3A on NC. Therefore, cell’s quiescent
state prior to plating is required for the consistency of the matrix model. Despite the need for
large quantities of protein for the subsequent IP experiment, one would have to starve the
transfected cells before the experiment.
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Figure 20: Plating without prior starvation induces nuclear DNMT3A expression
Transfected BSMCs were plated onto different matrices with or without prior serum starvation.
Cells were fixed at 48 hours and visualized with a myc-tag antibody (n=4). Nuclear DNMT3A
staining was observed in the non-starved cells.
Myc-DNMT3A exhibit consistent localization patter with endogenous DNMT3A
I conducted a timecourse experiment to confirm that the myc-DNMT3A shows similar
localization patterns with the endogenous DNMT3A, thus implying that the tagged DNMT3A is
regulated and trafficked in similar manner with its endogenous counterpart and could potentially
yield reliable IP results downstream. Briefly, myc-3A-BSMCs (48 hours transfection followed
by 24 hours of serum starvation) were plated onto NC or DNC and the cells were fixed at the 3
hour, 6 hour, 12 hour, 24 hour and 48 hour time points. A myc-tag and a DNMT3A primary
87
antibody were used to stain the cells. As shown in Figure 21 A and B, the endogenous and
plasmid expressed DNMT3A have the same patterns of localization at earlier time points on both
NC and DNC. Furthermore, the DNMT3A trafficking in transfected huBSMC is consistent with
the pattern observed in earlier timecourse experiment using non-transfected rat BSMC and
huBSMC, both at earlier timepoints as well as at 48 hours (Figure 21 C).
In summary, to transfect primary huBSMC with the myc-DNMT3A plasmid and to use the cells
for subsequent matrix experiment, cells (80% confluency) need to be transfected with 1.0 ug of
the myc-DNMT3A plasmid using the “P-024” program. After growing in culture for 48 hours
post transfection, myc-3A-BSMC needs to be serum starved to a quiescent state before plating
onto NC or DNC.
Immunoprecipitation and Mass Spectrometry (MS) requires large amount of
starting materials
Next, I isolated protein from myc-3A-BSMCs plated on NC and DNC. Proteins were extracted
from 6 x 106 cells (per sample) using the Nuclear Extraction kit (Active Motif) the IP was
performed using myc-tag agarose beads (Sigma) according to manufacturers’ protocols. A
protein band was detected in the nuclear fraction of cells plated on DNC (Figure 22).
The experiment was repeated with 1x 107 cells used for each IP reaction and the IP product was
submitted to the Mass Spectrometry facility at the Hospital for Sick Children. No significant
amount of protein was detected by the MS, including the bait protein myc-DNMT3A.
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Figure 21: Timecourse experiment using transfected human BSMC
Transfected BSMCs were plated onto different matrices and the cells were fixed at different
points and visualized by DNMT3A and myc-tag antibodies (n=4). At earlier timepoints (3, 6 and
12 hours), transfected cells showed similar patterns of DNMT3A localization on both NC (A)
and DNC (B) with respect to Figure 4 A. At 48 hours (C), transfected cells show consistent
DNMT3A localization pattern with non-transfected cells. Furthermore, the endogenous and myc-
tagged DNMT3A had the same subcellular localization at all timepoints.
Figure 22 : Myc-DNMT3A is detected in the IP product of transfected human BSMCs
Protein lysate (nuclear and cytosolic) from 6 x 106 transfected human BSMCs was
immunoprecipitated using a myc-tag antibody anchored on agrose beads (Sigma Aldrich). The IP
product was run on a western gel and was probed using a myc-tag primary antibody. A band was
observed in the nuclear fraction.
Discussion and future directions
Using the Nucleofector II system, I have successfully transfected primary huBSMCs with the
myc-DNMT3A plasmid. I have shown that increased expression of DNMT3A does not alter its
localization and that the plasmid expressed DNMT3A is trafficked in a consistent manner with
the endogenously expressed protein.
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Optimal transfection conditions
By varying the amount of plasmid per transfection and by monitoring the myc-DNMT3A
expression at different timepoints, I demonstrated that the highest transfection efficiencies occur
at 48 hours post transfection. Lower amount of plasmid used per reaction (0.5 ug) showed the
least level of plasmid toxicity, as seen by similar levels of cells per field at 24 and 72 hours.
However, the transfection efficiency was also considerably lower than the two other groups. On
the other hand, the 2.0 ug per reaction group had higher transfection efficiencies but high levels
of cell death at all three timepoints with a survival rate less than 25% compared to non-
transfected cells. The 1.0 ug per reaction was considered to be the optimal group because it had a
higher transfection efficiency (vs. 0.5 ug) and a lower rate of cell death (vs. 2.0 ug). Even though
the downstream IP experiment required large amounts of the protein bait (myc-DNMT3A) and
serum starvation decreases the plasmid expression; we have shown that serum starvation prior to
plating onto matrices is essential for the model’s consistency. The pathways related to the level
of mitosis and cell cycle regulation seem to predominantly regulate the expression level and
intracellular trafficking of DNMT3A. Cell cycle arrest, induced by Nacodazole, prevented the
nuclear localization of DNMT3A on DNC while the addition of FBS prior to plating induced
DNMT3A localization on NC. Two well-known SMC mitogens, EGF and bFGF, however, did
not alter the expression or trafficking of DNMT3A on different matrices.
Large amount of starting material is required for MS
Despite observing the myc-DNMT3A on a western gel, the MS did not detect any significant
amount of protein. This could be due to the fact that the signal was amplified by the exposure
time in the western experiment and, therefore, it overestimated the amount of protein eluted
from IP.
Due to the nature of our matrix model, we are extremely limited in the number of cells that can
be harvested on the matrices. Firstly, I have shown that serum depletion prior to plating on
matrices is required, which invariably reduces the expression of the bait protein. Second, as we
have shown, the DNMT3A localization pattern depends on a low cell density (Figure #), BSMCs
are plated at 5 x 104 cells/mL in our experiments. Each 10 cm dish (10 mL of media per plate),
coated with NC or DNC, would only yield 5 x 105 cells on NC at the 48 hour timepoint ( BSMCs
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are very quiescent on NC). Ultimately, up to 1 x 108 cells per IP reaction may be sufficient for
the MS identification experiment, one sample for the MS experiment needs about 200 plates of
cells plated on NC.
Future directions
The IP – MS experiment is limited by the expression of myc-DNMT3A bait protein. Since the in
vitro matrix model requires cell starvation and involves re-plating cells onto different matrices,
the amount of myc-3A protein may be significantly reduced by the end of the experiment.
Alternatively, one may try to select cells with stable expression of the plasmid using the
selection marker (treating the cells with geneticin for prolonged a period of time) or transduce
the plasmid using lentivirus.
Secondly, one may also try seeding the BSMCs into the collagen gel (3D) rather than plating
them on the surface of the gel (2D). This will help to reduce the amount of material as well as
time spent plating cells. For NC, the model would be most probably consistent as a significant
portion of cells migrate into the gel during the course of experiment. However, for DNC, one
needs to confirm whether plating into 3D gel instead of onto 2D gel (in our current model) will
alter BSMC phenotype and will alter DNMT3A localization pattern. Initially, we have shown
that a mixture of NC: DNC 3D gel still induced the nuclear upregulation of DNMT3A (Figure 2).
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Appendix II: Supplemental Figure and Table
Table 9: Gene expression changes from microarray analysis 124
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Figure 23: AKT signaling is upregulated on DNC at 3 hours.
Previously serum starved human BSMCs were plated onto NC or DNC and cells were isolated at
3 and 48 hours. Whole cell protein lysates were run on a western gel and probed with different
primary antibodies (listed on the left, all primary antibodies were purchased from Cell Signaling).
At 3 hours, both the AKT and the mTOR pathway is activated on DNC but not at 48 hours.
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Figure 24. CpG island upstream of the human KCNB2 gene
A CpG island (110 CpG sites) is present upstream of the human KCNB2 gene promoter140.
Transcription factor ChIP-seq data shows EZH2 binding overlapping with the CpG island188.
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