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

References

1. Aitken, K. University of Toronto (2011).

2. Campbell's Urology, (Saunders, New York, 2002).

3. Keane, D.P. & O'Sullivan, S. Urinary incontinence: anatomy, physiology and

pathophysiology. Baillieres Best Pract Res Clin Obstet Gynaecol 14, 207-26 (2000).

4. Fowler, C.J., Griffiths, D. & de Groat, W.C. The neural control of micturition. Nat Rev

Neurosci 9, 453-66 (2008).

5. Malmqvist, U., Arner, A. & Uvelius, B. Contractile and cytoskeletal proteins in smooth

muscle during hypertrophy and its reversal. Am J Physiol 260, C1085-93 (1991).

6. Austin, J.C., Chacko, S.K., DiSanto, M., Canning, D.A. & Zderic, S.A. A male murine

model of partial bladder outlet obstruction reveals changes in detrusor morphology,

contractility and Myosin isoform expression. J Urol 172, 1524-8 (2004).

7. Metcalfe, P.D. et al. Bladder outlet obstruction: progression from inflammation to

fibrosis. BJU Int 106, 1686-94 (2010).

8. Moore, C.K., Levendusky, M. & Longhurst, P.A. Relationship of mass of obstructed rat

bladders and responsiveness to adrenergic stimulation. J Urol 168, 1621-5 (2002).

9. Chen, M.W., Levin, R.M. & Buttyan, R. Peptide growth factors in normal and

hypertrophied bladder. World J Urol 13, 344-8 (1995).

10. Diwan, A. & Dorn, G.W., 2nd. Decompensation of cardiac hypertrophy: cellular

mechanisms and novel therapeutic targets. Physiology (Bethesda) 22, 56-64 (2007).

11. Kostin, S. et al. Connexin 43 expression and distribution in compensated and

decompensated cardiac hypertrophy in patients with aortic stenosis. Cardiovasc Res 62,

426-36 (2004).

12. Chang, S. et al. Alteration of the PKC-mediated signaling pathway for smooth muscle

contraction in obstruction-induced hypertrophy of the urinary bladder. Lab Invest 89,

823-32 (2009).

13. Ikeda, S., Hamada, M. & Hiwada, K. Contribution of non-cardiomyocyte apoptosis to

cardiac remodelling that occurs in the transition from compensated hypertrophy to heart

failure in spontaneously hypertensive rats. Clin Sci (Lond) 97, 239-46 (1999).

14. Bennett, M.R. Cell death in cardiovascular disease. Arterioscler Thromb Vasc Biol 31,

2779-80 (2011).

15. Weston, P.M., Robinson, L.Q., Williams, S., Thomas, M. & Stephenson, T.P. Poor

compliance early in filling in the neuropathic bladder. Br J Urol 63, 28-31 (1989).

65

16. Malkowicz, S.B. et al. Acute biochemical and functional alterations in the partially

obstructed rabbit urinary bladder. J Urol 136, 1324-9 (1986).

17. Ganz, M.L. et al. Economic costs of overactive bladder in the United States. Urology 75,

526-32, 532 e1-18 (2010).

18. Peters, C.A. & Bauer, S.B. Evaluation and management of urinary incontinence after

surgery for posterior urethral valves. Urol Clin North Am 17, 379-87 (1990).

19. Holmdahl, G. Bladder dysfunction in boys with posterior urethral valves. Scand J Urol

Nephrol Suppl 188, 1-36 (1997).

20. Rosenberg, M.T. The treatment of overactive bladder: a primary care provider's

perspective. Curr Urol Rep 9, 428-32 (2008).

21. Yamaguchi, T., Nagano, M. & Osada, Y. Effects of different alpha-1 adrenoceptor

blockers on proximal urethral function using in vivo isovolumetric pressure changes. J

Smooth Muscle Res 41, 247-56 (2005).

22. Karsenty, G. et al. Botulinum toxin A (Botox) intradetrusor injections in adults with

neurogenic detrusor overactivity/neurogenic overactive bladder: a systematic literature

review. Eur Urol 53, 275-87 (2008).

23. Hanna-Mitchell, A.T., Kashyap, M., Chan, W.V., Andersson, K.E. & Tannenbaum, C.

Pathophysiology of idiopathic overactive bladder and the success of treatment: a

systematic review from ICI-RS 2013. Neurourol Urodyn 33, 611-7 (2014).

24. Aitken, K.J. et al. Mammalian target of rapamycin (mTOR) induces proliferation and de-

differentiation responses to three coordinate pathophysiologic stimuli (mechanical strain,

hypoxia, and extracellular matrix remodeling) in rat bladder smooth muscle. Am J Pathol

176, 304-19 (2010).

25. Weichhart, T. Mammalian target of rapamycin: a signaling kinase for every aspect of

cellular life. Methods Mol Biol 821, 1-14 (2012).

26. McMullen, J.R. et al. Inhibition of mTOR signaling with rapamycin regresses established

cardiac hypertrophy induced by pressure overload. Circulation 109, 3050-5 (2004).

27. Paddenberg, R. et al. Rapamycin attenuates hypoxia-induced pulmonary vascular

remodeling and right ventricular hypertrophy in mice. Respir Res 8, 15 (2007).

28. Schroder, A., Kirwan, T.P., Jiang, J.X., Aitken, K.J. & Bagli, D.J. Rapamycin attenuates

bladder hypertrophy during long-term outlet obstruction in vivo: tissue, matrix and

mechanistic insights. J Urol 189, 2377-84 (2013).

29. Stein, R. et al. The decompensated detrusor V: molecular correlates of bladder function

after reversal of experimental outlet obstruction. J Urol 166, 651-7 (2001).

66

30. Guan, Z., Kiruluta, G., Coolsaet, B. & Elhilali, M. A minipig model for urodynamic

evaluation of infravesical obstruction and its possible reversibility. J Urol 154, 580-6

(1995).

31. Gabella, G. & Uvelius, B. Reversal of muscle hypertrophy in the rat urinary bladder after

removal of urethral obstruction. Cell Tissue Res 277, 333-9 (1994).

32. Malmqvist, U., Arner, A. & Uvelius, B. Cytoskeletal and contractile proteins in detrusor

smooth muscle from bladders with outlet obstruction--a comparative study in rat and man.

Scand J Urol Nephrol 25, 261-7 (1991).

33. Uvelius, B., Lindner, P. & Mattiasson, A. Collagen content in the rat urinary bladder

following removal of an experimental infravesical outlet obstruction. Urol Int 47, 245-9

(1991).

34. Wolffenbuttel, K.P., de Jong, B.W., Scheepe, J.R. & Kok, D.J. Potential for recovery in

bladder function after removing a urethral obstruction. Neurourol Urodyn 27, 782-8

(2008).

35. Buttyan, R., Chen, M.W. & Levin, R.M. Animal models of bladder outlet obstruction and

molecular insights into the basis for the development of bladder dysfunction. Eur Urol 32

Suppl 1, 32-9 (1997).

36. Greenland, J.E. et al. The effect of bladder outlet obstruction on tissue oxygen tension

and blood flow in the pig bladder. BJU Int 85, 1109-14 (2000).

37. Christiaansen, C.E., Sun, Y., Hsu, Y.C. & Chai, T.C. Alterations in expression of HIF-

1alpha, HIF-2alpha, and VEGF by idiopathic overactive bladder urothelial cells during

stretch suggest role for hypoxia. Urology 77, 1266 e7-11 (2011).

38. Sabha, N. et al. Matrix metalloproteinase-7 and epidermal growth factor receptor mediate

hypoxia-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein

kinase activation and subsequent proliferation in bladder smooth muscle cells. In Vitro

Cell Dev Biol Anim 42, 124-33 (2006).

39. Aitken, K.J. et al. Mechanotransduction of extracellular signal-regulated kinases 1 and 2

mitogen-activated protein kinase activity in smooth muscle is dependent on the

extracellular matrix and regulated by matrix metalloproteinases. Am J Pathol 169, 459-70

(2006).

40. Herz, D.B., Aitken, K. & Bagli, D.J. Collagen directly stimulates bladder smooth muscle

cell growth in vitro: regulation by extracellular regulated mitogen activated protein

kinase. J Urol 170, 2072-6 (2003).

41. Drzewiecki, B.A. et al. Modulation of the hypoxic response following partial bladder

outlet obstruction. J Urol 188, 1549-54 (2012).

42. Halachmi, S. et al. Role of signal transducer and activator of transcription 3 (STAT3) in

stretch injury to bladder smooth muscle cells. Cell Tissue Res 326, 149-58 (2006).

67

43. Sellers, J.R. & Adelstein, R.S. The mechanism of regulation of smooth muscle myosin by

phosphorylation. Curr Top Cell Regul 27, 51-62 (1985).

44. Li, S., Sims, S., Jiao, Y., Chow, L.H. & Pickering, J.G. Evidence from a novel human

cell clone that adult vascular smooth muscle cells can convert reversibly between

noncontractile and contractile phenotypes. Circ Res 85, 338-48 (1999).

45. Alexander, M.R. & Owens, G.K. Epigenetic control of smooth muscle cell differentiation

and phenotypic switching in vascular development and disease. Annu Rev Physiol 74, 13-

40 (2012).

46. Johansen, F.E. & Prywes, R. Serum response factor: transcriptional regulation of genes

induced by growth factors and differentiation. Biochim Biophys Acta 1242, 1-10 (1995).

47. Miano, J.M. Serum response factor: toggling between disparate programs of gene

expression. J Mol Cell Cardiol 35, 577-93 (2003).

48. Chen, J., Kitchen, C.M., Streb, J.W. & Miano, J.M. Myocardin: a component of a

molecular switch for smooth muscle differentiation. J Mol Cell Cardiol 34, 1345-56

(2002).

49. McDonald, O.G., Wamhoff, B.R., Hoofnagle, M.H. & Owens, G.K. Control of SRF

binding to CArG box chromatin regulates smooth muscle gene expression in vivo. J Clin

Invest 116, 36-48 (2006).

50. Long, X., Bell, R.D., Gerthoffer, W.T., Zlokovic, B.V. & Miano, J.M. Myocardin is

sufficient for a smooth muscle-like contractile phenotype. Arterioscler Thromb Vasc Biol

28, 1505-10 (2008).

51. Liu, Y. et al. Kruppel-like factor 4 abrogates myocardin-induced activation of smooth

muscle gene expression. J Biol Chem 280, 9719-27 (2005).

52. Liu, Z.P., Wang, Z., Yanagisawa, H. & Olson, E.N. Phenotypic modulation of smooth

muscle cells through interaction of Foxo4 and myocardin. Dev Cell 9, 261-70 (2005).

53. Majesky, M.W. Decisions, decisions...SRF coactivators and smooth muscle myogenesis.

Circ Res 92, 824-6 (2003).

54. Wang, Z. et al. Myocardin and ternary complex factors compete for SRF to control

smooth muscle gene expression. Nature 428, 185-9 (2004).

55. Hendrix, J.A. et al. 5' CArG degeneracy in smooth muscle alpha-actin is required for

injury-induced gene suppression in vivo. J Clin Invest 115, 418-27 (2005).

56. Gomez, D. & Owens, G.K. Smooth muscle cell phenotypic switching in atherosclerosis.

Cardiovasc Res 95, 156-64 (2012).

57. Nguyen, A.T. et al. Smooth muscle cell plasticity: fact or fiction? Circ Res 112, 17-22

(2013).

68

58. Rong, J.X., Shapiro, M., Trogan, E. & Fisher, E.A. Transdifferentiation of mouse aortic

smooth muscle cells to a macrophage-like state after cholesterol loading. Proc Natl Acad

Sci U S A 100, 13531-6 (2003).

59. Liu, R., Leslie, K.L. & Martin, K.A. Epigenetic regulation of smooth muscle cell

plasticity. Biochim Biophys Acta (2014).

60. Ailawadi, G. et al. Smooth muscle phenotypic modulation is an early event in aortic

aneurysms. J Thorac Cardiovasc Surg 138, 1392-9 (2009).

61. Burkhard, F.C., Lemack, G.E., Zimmern, P.E., Lin, V.K. & McConnell, J.D. Contractile

protein expression in bladder smooth muscle is a marker of phenotypic modulation after

outlet obstruction in the rabbit model. J Urol 165, 963-7 (2001).

62. Barendrecht, M.M. et al. The effect of bladder outlet obstruction on alpha1- and beta-

adrenoceptor expression and function. Neurourol Urodyn 28, 349-55 (2009).

63. Sobue, K. & Sellers, J.R. Caldesmon, a novel regulatory protein in smooth muscle and

nonmuscle actomyosin systems. J Biol Chem 266, 12115-8 (1991).

64. Ueki, N., Sobue, K., Kanda, K., Hada, T. & Higashino, K. Expression of high and low

molecular weight caldesmons during phenotypic modulation of smooth muscle cells.

Proc Natl Acad Sci U S A 84, 9049-53 (1987).

65. Mechtersheimer, G., Barth, T., Quentmeier, A. & Moller, P. Differential expression of

beta 1 integrins in nonneoplastic smooth and striated muscle cells and in tumors derived

from these cells. Am J Pathol 144, 1172-82 (1994).

66. Owens, G.K. & Thompson, M.M. Developmental changes in isoactin expression in rat

aortic smooth muscle cells in vivo. Relationship between growth and cytodifferentiation.

J Biol Chem 261, 13373-80 (1986).

67. Fatigati, V. & Murphy, R.A. Actin and tropomyosin variants in smooth muscles.

Dependence on tissue type. J Biol Chem 259, 14383-8 (1984).

68. Matsumoto, S., Hanai, T., Ohnishi, N., Yamamoto, K. & Kurita, T. Bladder smooth

muscle cell phenotypic changes and implication of expression of contractile proteins

(especially caldesmon) in rats after partial outlet obstruction. Int J Urol 10, 339-45

(2003).

69. Yang, L., He, D.L., Wang, S., Cheng, H.P. & Wang, X.Y. Effect of long-term partial

bladder outlet obstruction on caldesmon isoforms and their correlation with contractile

function. Acta Pharmacol Sin 29, 600-5 (2008).

70. DiSanto, M.E. et al. Alteration in expression of myosin isoforms in detrusor smooth

muscle following bladder outlet obstruction. Am J Physiol Cell Physiol 285, C1397-410

(2003).

69

71. Jones, P.L., Jones, F.S., Zhou, B. & Rabinovitch, M. Induction of vascular smooth

muscle cell tenascin-C gene expression by denatured type I collagen is dependent upon a

beta3 integrin-mediated mitogen-activated protein kinase pathway and a 122-base pair

promoter element. J Cell Sci 112 ( Pt 4), 435-45 (1999).

72. Mackie, E.J. Molecules in focus: tenascin-C. Int J Biochem Cell Biol 29, 1133-7 (1997).

73. Taipale, J. & Keski-Oja, J. Growth factors in the extracellular matrix. FASEB J 11, 51-9

(1997).

74. Aszodi, A., Legate, K.R., Nakchbandi, I. & Fassler, R. What mouse mutants teach us

about extracellular matrix function. Annu Rev Cell Dev Biol 22, 591-621 (2006).

75. Streuli, C. Extracellular matrix remodelling and cellular differentiation. Curr Opin Cell

Biol 11, 634-40 (1999).

76. Sheetz, M.P. Cell control by membrane-cytoskeleton adhesion. Nat Rev Mol Cell Biol 2,

392-6 (2001).

77. Wiesner, S., Legate, K.R. & Fassler, R. Integrin-actin interactions. Cell Mol Life Sci 62,

1081-99 (2005).

78. Spencer, V.A., Xu, R. & Bissell, M.J. Gene expression in the third dimension: the ECM-

nucleus connection. J Mammary Gland Biol Neoplasia 15, 65-71 (2010).

79. Bissell, M.J., Hall, H.G. & Parry, G. How does the extracellular matrix direct gene

expression? J Theor Biol 99, 31-68 (1982).

80. Daniel, C.W. & Deome, K.B. Growth of Mouse Mammary Glands in Vivo after

Monolayer Culture. Science 149, 634-6 (1965).

81. Bhat, R. & Bissell, M.J. Of plasticity and specificity: dialectics of the microenvironment

and macroenvironment and the organ phenotype. Wiley Interdiscip Rev Dev Biol 3, 147-

63 (2014).

82. Boulanger, C.A., Mack, D.L., Booth, B.W. & Smith, G.H. Interaction with the mammary

microenvironment redirects spermatogenic cell fate in vivo. Proc Natl Acad Sci U S A

104, 3871-6 (2007).

83. Petersen, O.W., Ronnov-Jessen, L., Howlett, A.R. & Bissell, M.J. Interaction with

basement membrane serves to rapidly distinguish growth and differentiation pattern of

normal and malignant human breast epithelial cells. Proc Natl Acad Sci U S A 89, 9064-8

(1992).

84. Paszek, M.J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8,

241-54 (2005).

85. Wilson, C.B., Leopard, J., Cheresh, D.A. & Nakamura, R.M. Extracellular matrix and

integrin composition of the normal bladder wall. World J Urol 14 Suppl 1, S30-7 (1996).

70

86. Prockop, D.J. & Kivirikko, K.I. Collagens: molecular biology, diseases, and potentials

for therapy. Annu Rev Biochem 64, 403-34 (1995).

87. Chang, S.L., Howard, P.S., Koo, H.P. & Macarak, E.J. Role of type III collagen in

bladder filling. Neurourol Urodyn 17, 135-45 (1998).

88. Longhurst, P.A., Eika, B., Leggett, R.E. & Levin, R.M. Urinary bladder function in the

tight-skin mouse. J Urol 148, 1611-4 (1992).

89. Aitken, K.J. & Bagli, D.J. The bladder extracellular matrix. Part I: architecture,

development and disease. Nat Rev Urol 6, 596-611 (2009).

90. Stevenson, K., Kucich, U., Whitbeck, C., Levin, R.M. & Howard, P.S. Functional

changes in bladder tissue from type III collagen-deficient mice. Mol Cell Biochem 283,

107-14 (2006).

91. Hinek, A. & Rabinovitch, M. 67-kD elastin-binding protein is a protective "companion"

of extracellular insoluble elastin and intracellular tropoelastin. J Cell Biol 126, 563-74

(1994).

92. Mithieux, S.M. & Weiss, A.S. Elastin. Adv Protein Chem 70, 437-61 (2005).

93. Landau, E.H. et al. Loss of elasticity in dysfunctional bladders: urodynamic and

histochemical correlation. J Urol 152, 702-5 (1994).

94. Inaba, M. et al. Upregulation of heme oxygenase and collagen type III in the rat bladder

after partial bladder outlet obstruction. Urol Int 78, 270-7 (2007).

95. Ewalt, D.H. et al. Is lamina propria matrix responsible for normal bladder compliance? J

Urol 148, 544-9 (1992).

96. Tekgul, S. et al. Collagen types I and III localization by in situ hybridization and

immunohistochemistry in the partially obstructed young rabbit bladder. J Urol 156, 582-6

(1996).

97. Deveaud, C.M. et al. Molecular analysis of collagens in bladder fibrosis. J Urol 160,

1518-27 (1998).

98. Baskin, L., Howard, P.S. & Macarak, E. Effect of physical forces on bladder smooth

muscle and urothelium. J Urol 150, 601-7 (1993).

99. Kaplan, E.P., Richier, J.C., Howard, P.S., Ewalt, D.H. & Lin, V.K. Type III collagen

messenger RNA is modulated in non-compliant human bladder tissue. J Urol 157, 2366-9

(1997).

100. Sutherland, R.S., Baskin, L.S., Elfman, F., Hayward, S.W. & Cunha, G.R. The role of

type IV collagenases in rat bladder development and obstruction. Pediatr Res 41, 430-4

(1997).

71

101. Cornut, P.L. et al. Microbiologic identification of bleb-related delayed-onset

endophthalmitis caused by moraxella species. J Glaucoma 17, 541-5 (2008).

102. Brancaccio, M. et al. Integrin signalling: the tug-of-war in heart hypertrophy. Cardiovasc

Res 70, 422-33 (2006).

103. Duncan, E.J., Gluckman, P.D. & Dearden, P.K. Epigenetics, plasticity, and evolution:

How do we link epigenetic change to phenotype? J Exp Zool B Mol Dev Evol 322, 208-

20 (2014).

104. Zhang, G. & Pradhan, S. Mammalian epigenetic mechanisms. IUBMB Life 66, 240-56

(2014).

105. Borsook, D. et al. Sex and the migraine brain. Neurobiol Dis 68, 200-14 (2014).

106. Sanchez, A.M. et al. The Endometriotic Tissue Lining the Internal Surface of

Endometrioma: Hormonal, Genetic, Epigenetic Status, and Gene Expression Profile.

Reprod Sci (2014).

107. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F. & Richmond, T.J. Crystal

structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251-60 (1997).

108. Peterson, C.L. & Laniel, M.A. Histones and histone modifications. Curr Biol 14, R546-

51 (2004).

109. Bell, O. et al. Accessibility of the Drosophila genome discriminates PcG repression,

H4K16 acetylation and replication timing. Nat Struct Mol Biol 17, 894-900 (2010).

110. Nguyen, C.T., Gonzales, F.A. & Jones, P.A. Altered chromatin structure associated with

methylation-induced gene silencing in cancer cells: correlation of accessibility,

methylation, MeCP2 binding and acetylation. Nucleic Acids Res 29, 4598-606 (2001).

111. Spitale, R.C., Tsai, M.C. & Chang, H.Y. RNA templating the epigenome: long noncoding

RNAs as molecular scaffolds. Epigenetics 6, 539-43 (2011).

112. Hung, T. & Chang, H.Y. Long noncoding RNA in genome regulation: prospects and

mechanisms. RNA Biol 7, 582-5 (2010).

113. Filipowicz, W., Bhattacharyya, S.N. & Sonenberg, N. Mechanisms of post-transcriptional

regulation by microRNAs: are the answers in sight? Nat Rev Genet 9, 102-14 (2008).

114. Reik, W. Stability and flexibility of epigenetic gene regulation in mammalian

development. Nature 447, 425-32 (2007).

115. Jurkowska, R.Z., Jurkowski, T.P. & Jeltsch, A. Structure and function of mammalian

DNA methyltransferases. Chembiochem 12, 206-22 (2011).

116. Takai, D. & Jones, P.A. Origins of bidirectional promoters: computational analyses of

intergenic distance in the human genome. Mol Biol Evol 21, 463-7 (2004).

72

117. Saxonov, S., Berg, P. & Brutlag, D.L. A genome-wide analysis of CpG dinucleotides in

the human genome distinguishes two distinct classes of promoters. Proc Natl Acad Sci U

S A 103, 1412-7 (2006).

118. Jinawath, A., Miyake, S., Yanagisawa, Y., Akiyama, Y. & Yuasa, Y. Transcriptional

regulation of the human DNA methyltransferase 3A and 3B genes by Sp3 and Sp1 zinc

finger proteins. Biochem J 385, 557-64 (2005).

119. Chen, Z.X., Mann, J.R., Hsieh, C.L., Riggs, A.D. & Chedin, F. Physical and functional

interactions between the human DNMT3L protein and members of the de novo

methyltransferase family. J Cell Biochem 95, 902-17 (2005).

120. Choi, S.H. et al. Identification of preferential target sites for human DNA

methyltransferases. Nucleic Acids Res 39, 104-18 (2011).

121. Lister, R. et al. Human DNA methylomes at base resolution show widespread

epigenomic differences. Nature 462, 315-22 (2009).

122. Deaton, R.A., Gan, Q. & Owens, G.K. Sp1-dependent activation of KLF4 is required for

PDGF-BB-induced phenotypic modulation of smooth muscle. Am J Physiol Heart Circ

Physiol 296, H1027-37 (2009).

123. Yoshida, T., Gan, Q., Shang, Y. & Owens, G.K. Platelet-derived growth factor-BB

represses smooth muscle cell marker genes via changes in binding of MKL factors and

histone deacetylases to their promoters. Am J Physiol Cell Physiol 292, C886-95 (2007).

124. Thomas, J.A. et al. PDGF-DD, a novel mediator of smooth muscle cell phenotypic

modulation, is upregulated in endothelial cells exposed to atherosclerosis-prone flow

patterns. Am J Physiol Heart Circ Physiol 296, H442-52 (2009).

125. Hodges, S.J., Yoo, J.J., Mishra, N. & Atala, A. The effect of epigenetic therapy on

congenital neurogenic bladders--a pilot study. Urology 75, 868-72 (2010).

126. Ekman, M. et al. Mir-29 repression in bladder outlet obstruction contributes to matrix

remodeling and altered stiffness. PLoS One 8, e82308 (2013).

127. Connelly, J.J. et al. Epigenetic regulation of COL15A1 in smooth muscle cell replicative

aging and atherosclerosis. Hum Mol Genet 22, 5107-20 (2013).

128. Ning, Y. et al. 5-Aza-2'-deoxycytidine inhibited PDGF-induced rat airway smooth

muscle cell phenotypic switching. Arch Toxicol 87, 871-81 (2013).

129. Mousa, A.A. et al. Preeclampsia is associated with alterations in DNA methylation of

genes involved in collagen metabolism. Am J Pathol 181, 1455-63 (2012).

130. Bhattacharya, S. & Schindler, C. Regulation of Stat3 nuclear export. J Clin Invest 111,

553-9 (2003).

131. Mal, A.K. Histone methyltransferase Suv39h1 represses MyoD-stimulated myogenic

differentiation. EMBO J 25, 3323-34 (2006).

73

132. Monte, E. et al. Systems proteomics of cardiac chromatin identifies nucleolin as a

regulator of growth and cellular plasticity in cardiomyocytes. Am J Physiol Heart Circ

Physiol 305, H1624-38 (2013).

133. Movassagh, M. et al. Differential DNA methylation correlates with differential

expression of angiogenic factors in human heart failure. PLoS One 5, e8564 (2010).

134. Haas, J. et al. Alterations in cardiac DNA methylation in human dilated cardiomyopathy.

EMBO Mol Med 5, 413-29 (2013).

135. Alexander, M.R., Murgai, M., Moehle, C.W. & Owens, G.K. Interleukin-1beta modulates

smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via

NF-kappaB-dependent mechanisms. Physiol Genomics 44, 417-29 (2012).

136. Azechi, T., Sato, F., Sudo, R. & Wachi, H. 5-aza-2'-Deoxycytidine, a DNA

methyltransferase inhibitor, facilitates the inorganic phosphorus-induced mineralization

of vascular smooth muscle cells. J Atheroscler Thromb 21, 463-76 (2014).

137. Jiang, J.X. et al. Phenotypic switching induced by damaged matrix is associated with

DNA methyltransferase 3A (DNMT3A) activity and nuclear localization in smooth

muscle cells (SMC). PLoS One 8, e69089 (2013).

138. Watson, C.J. et al. Hypoxia-induced epigenetic modifications are associated with cardiac

tissue fibrosis and the development of a myofibroblast-like phenotype. Hum Mol Genet

23, 2176-88 (2014).

139. Aapola, U., Liiv, I. & Peterson, P. Imprinting regulator DNMT3L is a transcriptional

repressor associated with histone deacetylase activity. Nucleic Acids Res 30, 3602-8

(2002).

140. Gardiner-Garden, M. & Frommer, M. CpG islands in vertebrate genomes. J Mol Biol 196,

261-82 (1987).

141. Lee, J.L., Wang, M.J. & Chen, J.Y. Acetylation and activation of STAT3 mediated by

nuclear translocation of CD44. J Cell Biol 185, 949-57 (2009).

142. di Bari, M.G. et al. c-Abl acetylation by histone acetyltransferases regulates its nuclear-

cytoplasmic localization. EMBO Rep 7, 727-33 (2006).

143. Pickard, A., Wong, P.P. & McCance, D.J. Acetylation of Rb by PCAF is required for

nuclear localization and keratinocyte differentiation. J Cell Sci 123, 3718-26 (2010).

144. Bonaldi, T. et al. Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it

towards secretion. EMBO J 22, 5551-60 (2003).

145. Chen, T., Ueda, Y., Xie, S. & Li, E. A novel Dnmt3a isoform produced from an

alternative promoter localizes to euchromatin and its expression correlates with active de

novo methylation. J Biol Chem 277, 38746-54 (2002).

74

146. Oh, B.K. et al. DNA methyltransferase expression and DNA methylation in human

hepatocellular carcinoma and their clinicopathological correlation. Int J Mol Med 20, 65-

73 (2007).

147. Liu, I. Bartlett Award, 2007. Confessions of a misfit. Theriogenology 68, 296-7 (2007).

148. Johnson-Saliba, M., Siddon, N.A., Clarkson, M.J., Tremethick, D.J. & Jans, D.A. Distinct

importin recognition properties of histones and chromatin assembly factors. FEBS Lett

467, 169-74 (2000).

149. Mosammaparast, N., Guo, Y., Shabanowitz, J., Hunt, D.F. & Pemberton, L.F. Pathways

mediating the nuclear import of histones H3 and H4 in yeast. J Biol Chem 277, 862-8

(2002).

150. Tolg, C. et al. Uropathogenic E. coli infection provokes epigenetic downregulation of

CDKN2A (p16INK4A) in uroepithelial cells. Lab Invest 91, 825-36 (2011).

151. Du, Z. et al. DNMT1 stability is regulated by proteins coordinating deubiquitination and

acetylation-driven ubiquitination. Sci Signal 3, ra80 (2010).

152. Zhou, Q., Agoston, A.T., Atadja, P., Nelson, W.G. & Davidson, N.E. Inhibition of

histone deacetylases promotes ubiquitin-dependent proteasomal degradation of DNA

methyltransferase 1 in human breast cancer cells. Mol Cancer Res 6, 873-83 (2008).

153. Sun, L. et al. Phosphatidylinositol 3-kinase/protein kinase B pathway stabilizes DNA

methyltransferase I protein and maintains DNA methylation. Cell Signal 19, 2255-63

(2007).

154. Ghoshal, K. et al. 5-Aza-deoxycytidine induces selective degradation of DNA

methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-

adjacent homology domain, and nuclear localization signal. Mol Cell Biol 25, 4727-41

(2005).

155. Kim, J.Y. & Casaccia, P. HDAC1 in axonal degeneration: A matter of subcellular

localization. Cell Cycle 9, 3680-4 (2010).

156. Sontag, R.L. & Weber, T.J. Ectopic ERK expression induces phenotypic conversion of

C10 cells and alters DNA methyltransferase expression. BMC Res Notes 5, 217 (2012).

157. Cowan, D.B. et al. Hypoxia and stretch regulate intercellular communication in vascular

smooth muscle cells through reactive oxygen species formation. Arterioscler Thromb

Vasc Biol 23, 1754-60 (2003).

158. Robert, L. & Labat-Robert, J. Aging of connective tissues: from genetic to epigenetic

mechanisms. Biogerontology 1, 123-31 (2000).

159. Ghafar, M.A. et al. Hypoxia and an angiogenic response in the partially obstructed rat

bladder. Lab Invest 82, 903-9 (2002).

75

160. Ghafar, M.A. et al. Effects of chronic partial outlet obstruction on blood flow and

oxygenation of the rat bladder. J Urol 167, 1508-12 (2002).

161. Stanzel, R.D., Lourenssen, S., Nair, D.G. & Blennerhassett, M.G. Mitogenic factors

promoting intestinal smooth muscle cell proliferation. Am J Physiol Cell Physiol 299,

C805-17 (2010).

162. Siddiqui, S. et al. The modulation of large airway smooth muscle phenotype and effects

of epidermal growth factor receptor inhibition in the repeatedly allergen-challenged rat.

Am J Physiol Lung Cell Mol Physiol 304, L853-62 (2013).

163. Falcon, B.L. et al. An in vitro cord formation assay identifies unique vascular phenotypes

associated with angiogenic growth factors. PLoS One 9, e106901 (2014).

164. Jackson, W.F. Potassium channels in the peripheral microcirculation. Microcirculation

12, 113-27 (2005).

165. Ng, D.H., Humphries, J.D., Byron, A., Millon-Fremillon, A. & Humphries, M.J.

Microtubule-dependent modulation of adhesion complex composition. PLoS One 9,

e115213 (2014).

166. Spin, J.M., Quertermous, T. & Tsao, P.S. Chromatin remodeling pathways in smooth

muscle cell differentiation, and evidence for an integral role for p300. PLoS One 5,

e14301 (2010).

167. Zhang, X., Mu, W. & Zhang, W. On the analysis of the illumina 450k array data: probes

ambiguously mapped to the human genome. Front Genet 3, 73 (2012).

168. Roessler, J. et al. Quantitative cross-validation and content analysis of the 450k DNA

methylation array from Illumina, Inc. BMC Res Notes 5, 210 (2012).

169. Movassagh, M. et al. Distinct epigenomic features in end-stage failing human hearts.

Circulation 124, 2411-22 (2011).

170. Sandoval, J. et al. Validation of a DNA methylation microarray for 450,000 CpG sites in

the human genome. Epigenetics 6, 692-702 (2011).

171. Banerjee, P., Suguna, L. & Shanthi, C. Wound healing activity of a collagen-derived

cryptic peptide. Amino Acids (2014).

172. Riopel, M., Stuart, W. & Wang, R. Fibrin improves beta (INS-1) cell function,

proliferation and survival through integrin alphavbeta3. Acta Biomater 9, 8140-8 (2013).

173. Zheng, D.Q., Woodard, A.S., Tallini, G. & Languino, L.R. Substrate specificity of

alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT

pathway activation. J Biol Chem 275, 24565-74 (2000).

174. Naga Prasad, S.V., Esposito, G., Mao, L., Koch, W.J. & Rockman, H.A. Gbetagamma-

dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload

hypertrophy. J Biol Chem 275, 4693-8 (2000).

76

175. Zuo, T. et al. Epigenetic silencing mediated through activated PI3K/AKT signaling in

breast cancer. Cancer Res 71, 1752-62 (2011).

176. Liu, Y., Yu, H. & Nimer, S.D. PI3K-Akt pathway regulates polycomb group protein and

stem cell maintenance. Cell Cycle 12, 199-200 (2013).

177. Cha, T.L. et al. Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine

27 in histone H3. Science 310, 306-10 (2005).

178. Elkelini, M.S., Aitken, K., Bagli, D.J. & Hassouna, M.M. Effects of doxycycline on

voiding behaviour of rats with bladder outlet obstruction. BJU Int 103, 537-40 (2009).

179. Iguchi, N., Hou, A., Koul, H.K. & Wilcox, D.T. Partial bladder outlet obstruction in mice

may cause E-cadherin repression through hypoxia induced pathway. J Urol 192, 964-72

(2014).

180. Ekman, M., Uvelius, B., Albinsson, S. & Sward, K. HIF-mediated metabolic switching in

bladder outlet obstruction mitigates the relaxing effect of mitochondrial inhibition. Lab

Invest 94, 557-68 (2014).

181. Semenza, G.L. Hydroxylation of HIF-1: oxygen sensing at the molecular level.

Physiology (Bethesda) 19, 176-82 (2004).

182. Parsons, B.A. & Drake, M.J. Animal models in overactive bladder research. Handb Exp

Pharmacol, 15-43 (2011).

183. Mourad, R. et al. Estrogen induces global reorganization of chromatin structure in human

breast cancer cells. PLoS One 9, e113354 (2014).

184. Blei, T. et al. Dose-dependent effects of isoflavone exposure during early lifetime on the

rat mammary gland: Studies on estrogen sensitivity, isoflavone metabolism, and DNA

methylation. Mol Nutr Food Res (2014).

185. de Conti, A. et al. Genotoxic, epigenetic, and transcriptomic effects of tamoxifen in

mouse liver. Toxicology 325, 12-20 (2014).

186. Kim, G.H., Ryan, J.J. & Archer, S.L. The role of redox signaling in epigenetics and

cardiovascular disease. Antioxid Redox Signal 18, 1920-36 (2013).

187. Sciarretta, S., Volpe, M. & Sadoshima, J. Mammalian target of rapamycin signaling in

cardiac physiology and disease. Circ Res 114, 549-64 (2014).

188. Gerstein, M.B. et al. Architecture of the human regulatory network derived from

ENCODE data. Nature 489, 91-100 (2012).

77

78

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

79

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).

80

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.

81

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).

82

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.

83

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.

84

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.

86

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.

88

89

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.

90

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

91

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).

92

Appendix II: Supplemental Figure and Table

Table 9: Gene expression changes from microarray analysis 124

93

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.

94

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.