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CALCIUM-SENSITIVE MECHANISMS IN VASCULAR SMOOTH MUSCLE CELL

CYCLE PROGRESSION AS TARGETS FOR THERAPY

by

Sonya K. Hui

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Physiology

University of Toronto

© Copyright by Sonya K. Hui, 2010

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ABSTRACT

Calcium-sensitive mechanisms in vascular smooth muscle cell cycle progression as targets for therapy Sonya K. Hui Master of Science Department of Physiology University of Toronto 2010

Increased intracellular calcium (Ca2+) is required for vascular smooth muscle cell (VSMC)

proliferation through mechanisms that are not well-known. Preventing calmodulin (CaM)-cyclin E

interaction with a synthetic peptide inhibits VSMC proliferation in a cyclin E-dependent manner, without

increasing de-differentiation or cell death, or affecting re-endothelialization or collagen deposition.

Moreover, in situ Ca2+-sensitive phosphorylation and degradation of the cell cycle inhibitor p27Kip1

(p27) in VSMC is specific to G1 and dependent on camodulin kinase-II (CaMK-II) and the proteasome,

but not MEK. Lastly, IQGAP1 binding to CaM increases during G1 with no change in total IQGAP1

expression across the cell cycle. Therefore, we determined the clinical potential of an established

mechanism (CaM/cyclin E), the existence of a putative mechanism (CaMK-II/p27), and a target novel

mechanism (CaM-IQGAP1). Characterization of calcium-sensitive mechanisms of VSMC cycle control

could form the basis for new drug-eluting stent agents that have increased selectivity for rapidly dividing

VSMC.

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ACKNOWLEDGEMENTS Pursuing this degree has both challenged and rewarded me in ways I did not anticipate. Although the end goal remains the same, what started off as a temporary detour has completely changed my path, and I am better because of it. Thank you to the individuals who have made this possible.

To Dr. Mansoor Husain, for taking a chance on me, supporting my aspirations, and having high expectations. Thank you for challenging me, I’ve achieved much more than I thought I could because you did. In learning from you, I have gone from reluctant to compelled to strive for a future in science. I am truly grateful for every opportunity you have given me and how it has impacted my career.

To Dr. Scott Heximer and Dr. Rod Bremner, thank you for being such patient, fair and sympathetic supervisors. I sincerely appreciate the time and thought you have dedicated to advising me and guiding my project.

To the Husain lab, for putting up with me on a daily basis. I appreciate every question that was answered and resource that was shared. Thank you for working with me, Dr. Talat Afroze, Dr. Hossein Noyan Ashraf, Dr. Ali Azam, Dr. Masayoshi Ishida, Dr. Sarah Steinbach, Dr. May Khalili, Dr. Omar El-Mounayri, Dr. Haiyan Xiao, Xingling Huang and

Tracey Richards, for taking care of numerous things so I wouldn’t have to worry about it. I was very spoiled to have your help. Thank you for letting me constantly interrupt you, and for being so thoughtful, considerate and sane.

Erin Mueller and Dr. Kiwon Ban, for looking out for me, tolerating my antics, and being like family in the lab.

Dr. Judith Hoefer, for going out of your way to try to rescue me, and everything it has led to. Thank you for your valuable insight and advice, and it has helped me immensely.

Dr. Syed Hassan Zaidi, for putting in extra time to help me work through things when I was stuck. Thank you for patiently explaining things to me, for your wise suggestions and for vouching for me. I sincerely appreciate your kindness and support.

Dr. Abdul Momen, for always greeting me with a smile. You are a delight to work with and a wonderful person.

To Dr. Steffen Bolz, for teaching the best course I’ve taken in years, and being an approachable, unique and inspiring mentor. Thank you for your objective, compassionate advice.

To our friendly neighbours and collaborators: the Liu, Li, Waddell, von Harsdorf, Bolz and Heximer labs, thank you for the favours and smiles in the hallway.

Thank you to those I have befriended along the way, who have pleasantly surprised, motivated and inspired me.

To Charlene Antony, for being a close companion and a genuinely sweet and caring person.

To Meghan Sauvé, for valiantly wanting to save me when I was in trouble, whether it be from broken cell phones, public speaking engagements or projectile rodents.

To Dr. Maral Ouzounian, for being clutch and coming through several times. You’re my hero! Thank you for caring about my goals, your encouragement has meant a lot to me.

To Shaan Chugh, for trying to keep me on track. Thank you for being consistent. And for consistently being ridiculous so I can laugh at you.

To Dr. Anja Meissner, for selflessly cheering me on with my pursuits, both inside and outside the lab. Thank you for your genuine interest in my project and lending your scientific expertise. I am so appreciative of your unwavering enthusiasm and support.

To Geoff de Couto, for being a pleasure to work with, and even better to hang out with.

To Anton Mihic and Dr. Keith Brunt, for setting an impressive example (most of the time), and showing me how great research can be. Thank you for being so generous with your time, and for sharing your perspective, intelligence and experience with me- it’s helped me pull things off and figure things out more than you know. The past year has been more productive, but also much more fun. I would love to work with you both any day.

To Susannah Moore, for lending me the projector and responding to my email(s). Thank you for being so welcoming, awesome and hilarious.

I’m grateful as always to my family and friends, for dealing with my frustrations and celebrating my successes, for checking in, picking me up and bringing me food. Thank you for providing balance, understanding when I wasn’t available, and for your constant, unconditional support.

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TABLE OF CONTENTS

ABSTRACT ……………………………………………………………………………………………… ii

ACKNOWLEDGMENTS ……………………………………………………………………………… iii

TABLE OF CONTENTS …………………………………………………………………………….… iv

LIST OF ABBREVIATIONS ………………….……………………………………………………..… x

LIST OF FIGURES AND TABLES …………………………………………………………………… xi

LIST OF APPENDICES ………………………………………………………………………………. xii

1.0 GENERAL INTRODUCTION ………………………………………………………………… 1

1.1 Background

1.1.1 Prevalence of cardiovascular disease

1.1.2 Unregulated smooth muscle cell proliferation and vascular pathologies

1.1.3 Targeting of cell cycle regulation as therapeutic strategy for treatment of proliferative vascular

diseases

1.1.4 Calcium/calmodulin signalling and regulation of cell cycle

1.1.5 Calicum/calmodulin-sensitive cell cycle regulation in vascular smooth muscle cells

1.2 Rationale ………………………………………………………………………………………… 3

1.2.1 Need for improved drug-eluting stent agents

1.2.2 Calmodulin/cyclin E interaction in vascular smooth muscle cells

1.3 Objectives ……………………………………………………………………………………...… 4

1.3.1 Clinical potential of established calcium-sensitive mechanisms (CBS)

1.3.2 Determination of novel calcium-sensitive mechanisms of cell cycle control

2.0 CLINICAL POTENTIAL OF A PEPTIDE INHIBITING CALMODULIN-CYCLIN E

INTERACTION IN VASCULAR SMOOTH MUSCLE …………………………………………...… 5

2.1 Authorship ………………………………………………………………………………………. 6

2.2 Abstract ………………………………………………………………………………………….. 7

2.3 Introduction ………………………………………………………………………………………8

2.3.1 Pathological vascular smooth muscle cell proliferation

2.3.2 Calcium-dependent regulation of vascular smooth muscle cell proliferation

2.3.3 Calmodulin/cyclin E interaction

2.4 Rationale ………………………………………………………………………………………… 8

2.5 Objectives ……………………………………………………………………………...………… 9

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2.6 Hypotheses …………………………………………………………………………………….… 9

2.7 Materials and Methods ………………………………………………………….……………… 9

2.7.1 Cell culture

2.7.2 Generation of synthetic peptides

2.7.3 TAT-mediated peptide delivery

2.7.4 Cell counting

2.7.5 Tritiated-thymidine incorporation assay

2.7.6 LDH assay

2.7.7 Caspase-3 assay

2.7.8 TUNEL staining assay

2.7.9 Mouse carotid artery injury

2.7.10 Pluronic gel administration of TAT-CBS

2.7.11 BrdU administration

2.7.12 Tissue processing and histology

2.7.13 Morphometry analysis

2.7.14 Collagen analysis

2.7.15 Immunohistochemistry staining

2.7.16 Immunofluorescent staining

2.7.17 Statistical Analysis

2.8 Results …………………………………………………………………………………………15

2.8.1 Modification of CBS peptide to increase bioavailability

2.8.2 TAT-CBS decreases cell number of human aortic smooth muscle cells as measured by cell

counting

2.8.3 TAT-CBS decreases proliferation of human aortic smooth muscle cells as measured by 3H-

thymidine incorporation

2.8.4 TAT-CBS decreases proliferation of human aortic endothelial cells

2.8.5 Anti-proliferative effect of TAT-CBS is dependent on cyclin E

2.8.6 TAT-CBS does not increase cytotoxcity in human aortic smooth muscle cells

2.8.7 TAT-CBS does not alter differentiation of human aortic smooth muscle cells as measured by

contractile smooth muscle cell marker immunofluorescent staining

2.8.8 TAT-CBS appears to have greater transduction efficiency in human aortic smooth muscle vs.

endothelial cells as indicated by His-tag immunostaining

2.8.9 Pluronic gel administration of TAT-CBS in vivo causes peptide delivery into smooth muscle cells

of the arterial wall post-carotid injury

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2.8.10 Pluronic gel administration of TAT-CBS in vivo decreases thickness of the intima

2.8.11 Pluronic gel administration of TAT-CBS in vivo decreases increases expression of smooth muscle

22-α

BrdU

2.8.12 Pluronic gel administration of TAT-CBS does not affect collagen deposition in vivo post-carotid

injury

2.8.13 Pluronic gel administration of TAT-CBS does not affect re-endothelialization in vivo post-carotid

injury

2.9 Discussion …………………………………………………………………………….………… 35

2.9.1 Summary

2.9.1.1 Previous findings with CBS peptide

2.9.1.2 TAT-CBS findings

2.9.2 Implications

2.9.2.1 Selectivity for calcium-sensitive, rapid, pathological proliferation

2.9.2.2 Selectivity for smooth muscle vs. endothelial cell transduction

2.9.3 Limitations

2.9.3.1 Potential non-specific TAT activity

2.9.3.2 Pluronic gel delivery

2.9.3.3 Wire carotid artery injury

2.9.3.4 Similar anti-proliferative effect in smooth muscle vs. endothelial cells

2.9.4 Future Directions

2.9.4.1 Cell cycle analysis with TAT-CBS

2.9.4.2 CBS as a novel drug-eluting stent agent: smooth muscle cell-selective delivery strategies

2.9.4.3 Promise of small molecule-based therapies

3.0 NOVEL CALCIUM-SENSITIVE MECHANISMS OF VASCULAR SMOOTH MUSCLE

CELL CYCLE CONTROL ………………………………………………………….………………… 42

3.1 Abstract ………………………………………………………………………………………… 43

3.2 Introduction ………………………………………………………………………………….… 44

3.2.1 Calcium-sensitive targets of cell cycle control

3.2.2 Putative mechanism of calcium-sensitive cell cycle control in vascular smooth muscle cells:

p27Kip1

3.2.3 Cell cycle inhibitor p27Kip1

3.2.3.1 Classic p27 function: cell cycle inhibition of cdk2

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3.2.3.2 P27 is “intrinsically unstructured”

3.2.4 Complexity of p27 regulation

3.2.4.1 Classic p27 degradation

3.2.4.2 Non-classical mechanisms of p27 degradation

3.2.5 Calcium signalling and p27 regulation

3.3 Rationale …………………………………………………………………….…………………. 46

3.3.1 Implication of p27 in inhibition of smooth muscle cell proliferation

3.3.2 Expression of p27 in proliferative vascular pathologies: restenosis and atherosclerosis

3.4 Objectives …………………………………………………………………………………….… 47

3.4.1 Investigation of putative calcium/calmodulin-sensitive p27 degradation

3.4.2 Novel calcium/calmodulin-sensitive mechanisms of cell cycle control

3.5 Hypothesis ……………………………………………………………………………………… 47

3.6 Materials and Methods ……………………………………………………………………...… 47

3.6.1 Cell culture

3.6.2 Cell cycle synchronization

3.6.3 In situ [Ca2+] manipulation

3.6.4 In situ inhibition of CaMKII, MEK and ubiquitin proteasome

3.6.5 Protein extraction

3.6.6 Calcium treatment of whole cell extracts

3.6.7 Immunoprecipitation

3.6.8 Western blot

3.6.9 Coomassie staining

3.6.10 Mass spectroscopy


3.7 Results: Role of calcium/calmodulin on p27 degradation in vascular smooth muscle

cells………………………………………………………………………………………………………. 50

3.7.1 Studies in whole cell protein extracts

3.7.2 Expression of p27 across the cell cycle in MOVAS

3.7.2.1 Characterization of cell cycle-dependent p27 degradation in MOVAS

3.7.2.2 Characterization of cell cycle-dependent p27 Thr 187 phosphorylation in MOVAS

3.7.3 In situ calcium analysis of p27 degradation in quiescent MOVAS

3.7.3.1 Increased intracellular calcium does not affect p27 degradation in G0-synchronized MOVAS

in situ

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3.7.3.2 Increased intracellular calcium does not affect Thr-187 phosphorylation of p27 in G0-

synchronized MOVAS in situ

3.7.4 In situ temporal analysis of calcium-sensitive p27 degradation in proliferating MOVAS

3.7.5 In situ cell cycle analysis of calcium-sensitive p27 degradation in proliferating MOVAS

3.7.6 In situ analysis of CaMKII/MEK/ubiquitin proteasome pathway of p27 degradation in

proliferating MOVAS

3.7.6.1 In situ analysis of CaMKII-sensitive p27 degradation in proliferating MOVAS

3.7.6.2 In situ analysis of MEK-sensitive p27 degradation in proliferating MOVAS

3.7.6.3 In situ analysis of ubiquitin proteasome-sensitive p27 degradation in proliferating MOVAS

3.8 Results: Identification of novel Ca2+/CaM-sensitive VSMC cycle proteins ………...……… 64

3.8.1 Broad survey of cell cycle differential calmodulin-binding proteins in MOVAS

3.8.2 Identification of IQGAP1 by mass spectroscopy

3.8.3 IQGAP1 is uniformly expressed across the cell cycle in MOVAS

3.9 Discussion ………………………………………………………………………………….…… 64

3.9.1 Summary

3.9.2 Limitations

3.9.2.1 Pharmacological inhibitors

3.9.2.2 Cell cycle synchronization of MOVAS

3.9.2.3 In vitro studies only


3.9.3.1 Further exploration of putative calcium-sensitive p27 degradation pathway

3.9.3.2 Increase quantitative resolution of analyses

3.9.3.3 Elucidation of remaining components of calcium-p27 pathway

3.9.3.4 Putative cell cycle involvement of IQGAP1

3.9.4 Implications

3.9.4.1 Adding another layer of understanding to complex p27 regulation

3.9.4.2 Targeting p27 degradation is an effective method of treating restenosis

3.9.4.2.1 Insufficiency of endogenous p27 activity in pathological smooth muscle cell proliferation

3.9.4.2.2 Examples of effective p27 targeting for in vivo inhibition of smooth muscle cell

proliferation

4.0 GENERAL DISCUSSION …………………………………………………………………..…71

4.1 Interpretation ………………………………………………………………………………...…71

4.1.1 Relationship of reported calcium-sensitive mechanisms in vascular smooth muscle cells

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4.1.2 Overall calcium handling in vascular smooth muscle cells

4.2 Limitations ……………………………………………………………………………………... 71

4.2.1 Potential calcium-sensitive cell cycle regulation in endothelial cells

4.2.2 Requirement of gene therapy approach for smooth muscle cell-specific delivery

4.2.3 Contribution of extracellular matrix, circulating progenitors to vascular disease pathologies

4.2.4 Cyclin E/cdk2-independent cell cycle progression

4.3 Implications/clinical significance ……………………………………………………………... 73

4.3.1 New generation of drug-eluting stent agents based on calcium-sensitive cell cycle mechanisms

4.3.2 CBS and similar agents as novel cancer therapy

4.4 Future Directions ……………………………………………………………………………… 73

4.4.1 Narrowing down essential motifs

4.4.2 Translational testing of CBS and similar agents

4.4.3 TAT-CBS in cancer

4.4.4 Investigation of other novel calcium-sensitive cell cycle mechanisms in vascular smooth muscle

cells

5.0 REFERENCES ………………………………………………………………………………… 75

6.0 APPENDIX …………………………………………………………………………………..… 84

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LIST OF ABBREVIATIONS

α-SMA alpha-smooth muscle actin

Ca2+ calcium

CaM calmodulin

CaMK-I calmodulin-dependent kinase I

CaMK-II calmodulin-dependent kinase-II

CBS calmodulin binding site of cyclin E

cdk cyclin-dependent kinase

cdki cyclin-dependent kinase inhibitor

Cyc E DKO cyclin E1/2 double-knockout

DES drug-eluting stents

EC endothelial cells

HA-EC human aortic endothelial cells

HA-SMC human aortic smooth muscle cells

KIP kinase inhibitor protein

MEF mouse embryonic fibroblasts

Mut Cyc E N-terminal deleted mutant form of cyclin E1

NFAT nuclear factor of activated T-cells

NOS nitric oxide synthase

P21 p21Cip1

P27 p27Kip1

P-p27 Thr187-phosphorylated p27

PCI percutaneous coronary intervention

PCNA proliferating cell nuclear antigen

PDE phosphodiesterase

PMCA1 plasma membrane calcium-ATPase 1

PPAR-δ peroxisome proliferator-activated receptor-delta

SERCA sarcoplasmic reticulum calcium-ATPase

Skp2 SCFSkp2

SM22-α smooth muscle 22-alpha

SMC smooth muscle cell

sm-MHC smooth muscle myosin heavy chain

VSMC vascular smooth muscle cells

WCE whole cell extracts

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LIST OF FIGURES AND TABLES

Figure 1 TAT-CBS-His peptide delivery in vitro …………………….…………………………. 16

Figure 2 Anti-proliferative effect of TAT-CBS in human aortic SMC measured by cell counting

……………………………………………………………………………………………………………..17

Figure 3 Anti-proliferative effect of TAT-CBS in human vascular cells as measured by 3H-

thymidine incorporation …………………………………………………………………………………. 18

Figure 4 Cyclin E–dependent anti-proliferative effects of TAT-CBS measured by 3H-thymidine

incorporation …………………………………………………………………………………………..… 20

Figure 5 TAT-CBS does not increase cell death in human aortic SMC ………………………… 21

Figure 6 TAT-CBS did not appreciably alter expression of smooth muscle cell markers in human

aortic SMC ………………………………………………………………………………………………. 24

Figure 7 TAT-CBS appears to have greater transduction efficiency in human aortic SMC than

EC……………………………………………………………………………………………………….... 25

Figure 8 TAT-CBS-His peptide delivery to SMC of an injured carotid artery ………………….. 27

Figure 9 TAT-CBS decreases neointimal formation post-carotid injury ………………………... 28

Figure 10 TAT-CBS treatment maintains SM22-α expression post-carotid injury ………………. 34

Figure 11 TAT-CBS does not affect collagen deposition post-carotid injury ……………………. 36

Figure 12 TAT-CBS-His does not affect re-endothelialization in vivo……………………………. 38

Figure 13 Expression of p27 across the cell cycle in MOVAS …………………………………… 51

Figure 14 Thr 187 phosphorylation of p27 across the cell cycle in MOVAS …………..………… 53

Figure 15 In situ analysis of calcium-sensitive p27 degradation in quiescent MOVAS …..……… 54

Figure 16 In situ analysis of calcium-sensitive p27 Thr 187 phosphorylation in quiescent

MOVAS…………………….…………………………………………………………………………..... 55

Figure 17 In situ temporal analysis of calcium-sensitive p27 degradation in proliferating

MOVAS………………………………….…………………………………………………………….… 56

Figure 18 In situ analysis of CaMKII-sensitive p27 degradation in proliferating MOVAS ……… 60

Figure 19 In situ analysis of MEK-sensitive p27 degradation in proliferating MOVAS ……….… 62

Figure 20 In situ analysis of ubiquitin proteasome-sensitive p27 degradation in proliferating

MOVAS …………………………………………………………………………………………….…… 63

Figure 21 Differentially expressed CaM-binding proteins between G0 and 4 h of serum-stimulation

in MOVAS …………………………………………………………………………………………….… 65

Figure 22 IQGAP1 expression across the cell cycle in MOVAS ………………………………… 66

Table 1 Amino acid sequences of synthetic peptides used ……………….…………………..… 10

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LIST OF APPENDICES

Appendix 1 CBS prevents serum-stimulated increase in cell number and S-phase entry in a cyclin E-

dependent manner ……………………………………………………………………………………….. 85

Appendix 2 TAT-CBS-His decreases vascular smooth muscle cell proliferation in vivo ……...…… 87

Appendix 3 Carotid artery injury BrdU immunostaining …………………………………………… 88

Appendix 4 CBS inhibits calcium-sensitive CDK2 activity in VSMC …………………………...… 89

Appendix 5 Increased calcium/calmodulin does not affect p27 levels in MOVAS whole cell

extracts…………………………………………………………………………………………………… 90

Appendix 6 Restoring calmodulin to CaM-depleted MOVAS whole cell extracts does not affect p27

levels …………………………………………………………………………………………………..… 91

Appendix 7 Increased calcium/calmodulin does not affect p27 levels over time in MOVAS whole cell

extracts ………………………………………………………………………………………………...… 92

Appendix 8 Increased calcium/calmodulin does not affect p27 levels in 4 h serum-synchronized

MOVAS whole cell extracts ………………………………………………………………………..…… 95

Appendix 9 Increased calcium/calmodulin does not affect p27 levels in 6 h serum-synchronized

MOVAS whole cell exacts ………………………………………………………………………….…… 96

Appendix 10 Increased calcium/calmodulin does not affect p27 levels in WCE from WT and Cyclin E

DKO MEF ……………………………………………………………………………………………..… 97

Appendix 11 In situ analysis of calcium-sensitive p27 degradation in proliferating MOVAS …….… 99

CHAPTER 1. GENERAL INTRODUCTION

1.1 Background 1.1.1 Prevalence of cardiovascular disease

As the leading cause of morbidity and mortality in both developed and developing nations,

cardiovascular disease has truly reached pandemic proportions, significantly impacting quality-of-life and

healthcare resources worldwide1, 2. The primary cause of cardiovascular death is atherosclerosis3, chronic

inflammation and thickening of the artery wall in response to lipoprotein accumulation, which can lead to

ischemic insults such as myocardial infarction or stroke4. Interestingly, therapeutic interventions aimed at

expanding occluded atherosclerotic arteries such as balloon angioplasty or stenting can paradoxically

injure the blood vessel wall, and often lead to restenosis: recurrent narrowing of the artery5, 6. Studies of

early percutaneous coronary interventions (PCI) revealed that as many as 30% of dilated arteries

underwent restenosis, often requiring repeat procedures7. Although intra-lumenal stents have reduced the

negative remodeling that accompanies angioplasty8, 9, in-stent stenosis remains a serious complication10,

11.

1.1.2 Unregulated smooth muscle cell proliferation and vascular pathologies

Vascular smooth muscle cells (VSMC) of the blood vessel wall usually proliferate at very low

levels, remaining in the quiescent (G0) phase of the cell cycle. However, in response to growth

stimulatory factors, VSMC are able to re-enter the cell cycle, transform from a contractile and

differentiated to a synthetic and “de-differentiated” phenotype, and cause subsequent negative arterial

remodeling. Therefore, the uncontrolled growth and division of VSMC in adult arteries is a significant

aspect of several vascular disorders. For instance, the pathology of atherosclerosis is dependent in large

part on the unregulated proliferation of VSMC12-14. Moreover, restenosis is characterized by rapid

proliferation and migration of VSMC and subsequent intimal hyperplaisia15. VSMC proliferation also

contributes to the development of conditions such as cardiac transplant vasculopathy16, 17 and vein bypass

failure18.

1.1.3 Targeting of cell cycle regulation as therapeutic strategy for treatment of proliferative vascular

diseases

Accordingly, a detailed understanding of the molecular mechanisms underlying vascular smooth

muscle cell cycle regulation is an important therapeutic aim of cardiovascular research. Efforts aimed at

elucidating the fundamental mechanisms that govern VSMC proliferation are highly relevant to

understanding proliferative vascular diseases and their consequences. Previous research aimed at

1

understanding the molecular and cellular basis of proliferative vasculopathies focused on upstream

pathways does not account for inherent redundancies present in biological signaling systems. Therefore,

focusing effort on improving understanding of the final common pathway of cell cycle progression in

VSMC may prove a more selective and effective therapeutic strategy. The ability of differentiated VSMC

to repeatedly re-enter the cell cycle is unique among mature myocytes. This property underlies the

phenotypic plasticity of VSMC and forms the basis of their pathogenic potential. Therefore, it is critical to

address molecular mechanisms involved in the development and differentiation of VSMC, as they may

improve our ability to identify and prevent the “de-differentiation” of VSMC in disease.

1.1.4 Calcium/calmodulin signalling and regulation of cell cycle

As controlled cell division is process that is essential to the existence and survival of multi-

cellular organisms, several levels of cell cycle regulation have co-evolved19-21. In response to

environmental signals such as growth factors and nutrients, dividing cells coordinate cell cycle

progression to a restriction point in G1. If the necessary external factors are not present for proper cell

division, cells will arrest in G1. In order to exit G1, growth-stimulatory signals induce phophorylation of

retinoblastoma protein and subsequent activation of the E2F transcription factor family, after which

replication may proceed independently from external cues21. A series of “checkpoints” functions as

second level of internal control by preventing the initiation of downstream events prior to the accurate

completion of upstream ones22. This ensures the proper sequence of biochemical events necessary for

further cell cycle progression. The molecular basis of this regulation rests on the sequential activation of

members of a family of serine-threonine-specific protein kinases that consist of regulatory and catalytic

subunits termed cyclins and cyclin-dependent kinases (cdk), respectively23. The kinase activity of each

cdk is (i) increased by threonine phosphorylation, (ii) inhibited by phosphorylation at other threonine and

tyrosine residues, and (iii) by cdk inhibitors (cdki), which operate through a variety of mechanisms23, 24.

Extensive literature has established that the universal second messenger calcium (Ca2+) is

strongly associated with cell cycle regulation (see Reviews25-28), predominantly during two phases: early

G1 and the G1/S boundary 29. Studies have shown depletion of extracellular Ca2+, extracellular Ca2+ influx

and intracellular Ca2+ stores inhibits DNA synthesis and prevents progression to S phase in multiple cell

types28. Calmodulin (CaM), the main eukaryotic Ca2+ sensor, is a small, acidic protein able to bind up to

four calcium ions and mediate calcium signalling. Accordingly, calmodulin’s involvement in cell cycle

regulation has also been established through findings such as cell cycle arrest in the presence of CaM-

inhibiting drugs30 and anti-CaM antibodies, and CaM-dependent cell cycle progression occurring in a

2

dose-dependent fashion31. These studies and others provide evidence that Ca2+/CaM signalling is an

absolute requirement for cell growth and proliferation.

However, the promiscuous nature of Ca2+ as a second messenger, and the fact that Ca2+ does not

function as an exclusive modulator of any of the above cell cycle components have made the

identification of specific critical Ca2+-mediated events extremely difficult27. While several lines of

evidence implicate Ca2+ as a signal for gene expression32-34, it is unlikely that the acute requirement of

elevated intracellular Ca2+ for G1/S transitions is based on either a transcriptional or translational effect.

Accordingly, studies have supported the existence of direct regulatory interactions between Ca2+ or

Ca2+/CaM and cell cycle machinery35-40.

1.1.5 Calcium/calmodulin-sensitive cell cycle regulation in vascular smooth muscle cells

Calcium-sensitive cell cycle regulation has been demonstrated specifically in vascular smooth

muscle41. It has been shown that coordinate increases in intracellular free calcium are tightly regulated

and critically required for VSMC growth and cell division42. For instance, depletion of Ca2+ stores in the

G1 phase of VSMC results in a profound G1 arrest that is not overcome until internal Ca2+ stores are

replenished43, 44. A study utilizing the sarcoplasmic reticulum calcium-ATPase (SERCA) inhibitor

thapsigargin to diminish intracellular calcium reveals that Ca2+-deficient G1 arrest may occur via ERK1/2

nuclear translocation and inhibition of cyclin D expression45. In addition to intracellular calcium being

necessary for VSMC proliferation, the expression of calcium-handling proteins such as SERCA and the

ryanodine receptor (Ryr) have been shown to fluctuate in correspondence with VSMC cycle

progression46. Moreover, modified expression of the proto-oncogene c-myb, a known regulator of Ca2+

pumps and channels, or direct manipulation of plasma membrane calcium-ATPase 1 (PMCA1) affects

both calcium handling and cell cycle progression in VSMC42, 47, providing further evidence for the

involvement of calcium signalling in VSMC cycle control. However, despite a large body of knowledge

correlating calcium-signalling to proliferation, specific mechanisms of Ca2+-dependent cell cycle

regulation have not yet been determined in VSMC.

1.2 Rationale

1.2.1 Need for improved drug-eluting stent agents

Drugs such as serolimus and paclitaxel that interrupt VSMC proliferation and techniques for the

elution of such drugs from deployed stents have resulted in a tremendous reduction in the rates of clinical

restensosis48, 49. While usage of drug-eluting stents (DES) and other procedures such as brachytherapy

limit the extent of in-stent restenosis50-53, they are not without serious complications. In addition to

3

unregulated VSMC proliferation, vessel wall injury also causes denudation of the endothelial lining,

compromising the normally anti-thrombotic surface and protective barrier between VSMC and potentially

harmful circulating factors. Therefore, re-endothelialization is a critical component of the healing process

post-injury. However, as DES inhibit VSMC proliferation through local release of high concentrations of

powerful cell toxins, they can also impair endothelial healing, leading to persistent endothelial

dysfunction manifesting as residual vasodilatory deficits and/or long-term risk of thrombosis54. There is a

significant need for improved DES agents that are more selective, less toxic and not pro-thrombotic for

clinical use. Alternative strategies for more cost-effective and less harmful treatments of conditions such

as restenosis remain of great interest to interventional cardiology. Therefore, elucidation of calcium-

sensitive mechanisms of cell cycle control in VSMC could form the basis for development of novel DES

agents, as well as other therapeutic applications.

1.2.2 Calmodulin/cyclin E interaction in vascular smooth muscle cells

Our lab has recently defined a Ca2+/CaM-sensitive mechanism in VSMC: the binding of

calmodulin to cyclin E/cdk2, and the requirement of this interaction for calcium-dependent cell cycle

progression of VSMC through the G1/S checkpoint55. Moreover, preliminary studies show that a synthetic

peptide based on the sequence of the Calmodulin-Binding Site on cyclin E (CBS peptide) is able to block

Ca2+-sensitive activation of cyclin E/cdk2 activity, and is effective in arresting cell cycle progression of

VSMC. These data demonstrate the promise of targeting Ca2+-sensitive mechanisms of cell cycle

regulation for the development of novel treatments for vascular proliferative diseases.

1.3 Objectives

1.3.1 Clinical potential of established calcium-sensitive mechanisms (CBS)

We aim to determine the physiological and therapeutic relevance of calcium-sensitive

mechanisms of cell cycle regulation in vascular smooth muscle cells. Specifically, we will evaluate the

clinical potential of established calcium-sensitive mechanisms by testing the effectiveness of the CBS

peptide as a novel therapeutic agent.

1.3.2 Determination of novel calcium-sensitive mechanisms of cell cycle control

Given successful detection of CaM-cyclin E interaction and promising preliminary data, we also

aim to investigate and elucidate putative novel calcium-sensitive mechanisms in VSMC.

4

CHAPTER 2. CLINICAL POTENTIAL OF A PEPTIDE INHIBITING CALMODULIN-CYCLIN

E INTERACTION IN VASCULAR SMOOTH MUSCLE

Sonya Hui, Syed Zaidi, Abdul Momen,

Sarah Steinbach, May Khalili, Kiwon Ban, Mansoor Husain

5

2.1 Authorship

Unless otherwise stated Sonya Hui designed and performed experiments, analyzed results, and

prepared data for publication. Dr. Syed Zaidi assisted with experimental design, manuscript preparation

and His-tag immunostaining. Dr. Abdul Momen performed carotid artery injury, administration of

peptides in pluronic gel, perfusion fixation and harvesting of carotid arteries on mice. Dr. Sarah Steinbach

assisted with confocal microscopy. Dr. May Khalili performed some of the paraffin-embedding,

sectioning and H and E staining of carotid arteries. Dr. Kiwon Ban performed LDH and Caspase-3 assays

on peptide-treated cells.

6

2.2 Abstract

Binding of Ca2+/calmodulin (CaM) to cyclin E/cdk2 is necessary for Ca2+-sensitive G1-to-S phase

progression in vascular smooth muscle cells (VSMC). A synthetic CaM-Binding Sequence (CBS) peptide

blocked CaM-cyclin E interactions, prevented activation of cdk2, and abrogated Ca2+-sensitive G1-to-S

transitions in VSMC. Treatment with CBS peptide conjugated to the viral TAT transduction domain

(TAT-CBS) decreased proliferation of mouse VSMC in vitro and in vivo. This study aimed to further

characterize efficacy and mechanisms of action of TAT-CBS treatment on human aortic (HA)-SMC

proliferation in vitro and in a mouse model of neointima formation in vivo.

TAT-CBS treatment decreased HA-SMC and endothelial cell (HA-EC) proliferation in vitro as

indicated by tritiated-thymidine incorporation 3 days post-treatment. To determine peptide transduction

efficiency and lifespan, HA-SMC and EC were treated with TAT-CBS conjugated to a His tag. His-

immunostaining revealed a lifespan of approximately 3 days, with greater transduction efficiency in HA-

SMC vs. EC. Compared to HA-SMC treated with a negative control peptide (NC), treatment with TAT-

CBS achieved a dose-dependent decrease in cell number without increases in LDH, Caspase-3 or TUNEL

evidence of cell death. Compared to untreated controls, TAT-CBS did not appreciably alter expression of

smooth muscle cell markers SM22α, SM-MHC or SMα-actin as assessed by immunofluorescent staining.

Finally, compared to NC-treated animals, in vivo application of TAT-CBS in pluronic gel to injured

mouse carotid arteries significantly decreased neointima formation without affecting re-

endothelialization, as assessed by CD31 immunostaining 7 days post-injury, or collagen deposition as

assessed by Picro-Sirius Red staining and polarized light microscopy 14 days post-injury.

Inhibition of CaM binding to cyclin E/cdk2 with TAT-CBS treatment produces a long-term effect

on proliferation that outlasts the peptide’s lifespan in HA-SMC. TAT-CBS peptide exerts its

antiproliferative action without increasing de-differentiation or cell death of SMC in vitro, or altering re-

endothelialization or collagen deposition in vivo. These data highlight TAT-CBS as a novel candidate for

the treatment of restenosis, and support broad therapeutic targeting of Ca2+-sensitive cell cycle control in

VSMC.

7

2.3 Introduction 2.3.1 Pathological vascular smooth muscle cell proliferation

Vascular smooth muscle cells (VSMC) normally proliferate at very low rates in the media of

adult arteries, remaining in the growth arrested (G0) phase of the cell cycle. A shift in the balance between

growth stimulatory and inhibitory factors can lead to cell cycle re-entry and transformation from

contractile and quiescent to proliferative and synthetic phenotypes. Thus activated, VSMC can remodel

the artery by altering the extracellular matrix, replicating in the media, and migrating to the intima to

undergo further cycles of proliferation. Indeed, unregulated proliferation of VSMC is a principal

mechanism underlying the pathogenesis of common vascular diseases, such as atherosclerosis and

restenosis56, 57.

2.3.2 Calcium-dependent regulation of vascular smooth muscle cell proliferation

Decades of work have implicated ionic calcium (Ca2+) as a regulator of eukaryotic cell cycle

progression28. In VSMC, we previously made three related discoveries regarding Ca2+-mediated cell cycle

regulation: (i) a coordinated increase in the free intracellular Ca2+ concentration is required for G1-to S

phase cell cycle transition42, 47; (ii) this occurs through cell cycle-associated expression and activation of

specific Ca2+ pumps and channels47, 58, 59; and (iii) is at least partly mediated by Ca2+/calmodulin (CaM)-

dependent cyclin E/CDK2 activity55.

2.3.3 Calmodulin/cyclin E interaction

Our findings suggest that Ca2+-sensitivity of the G1 to S phase cell cycle transition requires the

direct binding of the major Ca2+ signal transducer CaM to cyclin E, through a specific and highly

conserved CaM-binding motif in cyclin E. The functional importance of this motif was accentuated by the

observation that a cyclin E mutant lacking this motif was unable to produce Ca2+/CaM-stimulated activity

of CDK255. These data shed light on a mechanistic basis for Ca2+-sensitive cell cycle progression, and

predicted other possible Ca2+/CaM-sensitive cell cycle targets29.

2.4 Rationale

Based on the discovery of a functional CaM-cyclin E interaction, we hypothesized that blocking

CaM-cyclin E binding through the use of a synthetic peptide would inhibit Ca2+-sensitive G1-to-S phase

transitions and slow the proliferation of VSMC by competing with cyclin E for binding to CaM.

Preliminary studies show a synthetic peptide based on the amino acid sequence of the CaM-Binding Site

of cyclin E (CBS peptide) can inhibit CaM-cyclin E interaction and Ca2+-sensitive enhancement of cyclin

E/cdk2 activity. Application of CBS peptide also decreases mouse VSMC number in vitro and reduces

arterial wall thickness in a mouse model of carotid injury in vivo.

8

2.5 Objectives

These promising findings suggest CBS may be a candidate for development as a novel treatment

for proliferative vascular conditions such as atherosclerosis and in-stent restenosis, and merit further

evaluation of possible therapeutic application. We aim to establish the clinical significance and examine

the potential of the CBS peptide.

2.6 Hypotheses

Accordingly, we hypothesize that TAT-mediated transduction of the CBS peptide will decrease

proliferation of human vascular smooth muscle cells and endothelial cells in vitro, and application of

TAT-CBS peptide will decrease proliferation of smooth muscle cells of the intima and media in a mouse

model of carotid injury in vivo.

2.7 Materials and Methods

2.7.1 Cell culture

Cells and reagents for primary human aortic SMC culture were purchased from Invitrogen.

Human VSMC (C-007-5C) were grown in Medium 231 with Smooth Muscle Growth Supplement (S-

007-25) and 1% penicillin-streptomycin. Cells were serum-starved by culturing in Media 231 with 2%

SGMS. Cells and reagents for primary human aortic endothelial cell (HA-EC) culture were purchased

from Invitrogen. HA-EC (C-006-5C) were grown in Medium 200 with Low Serum Growth Supplement

(S-003-10) and 1% penicillin-streptomycin (Invitrogen). Primary human cells used were below passage

10. Wild-type and cyclin E1/E2 double knockout (Cyc E DKO) mouse embryonic fibroblasts (MEF)

were kindly provided by Dr. P. Sicinski (Harvard Medical School), and maintained in DMEM with 10%

FBS (Hyclone) and 1% penicillin-streptomycin. G0 arrest of MEF was achieved by starvation for 48 h in

medium lacking FBS. All MEF used were under passage 4, as after passage 4 Cyc E DKO MEF undergo

a “replicative crisis”60.

2.7.2 Generation of synthetic peptides

All synthetic peptides (TAT-CBS-His, TAT-NC-His, TAT-CBS, TAT-NC and TAT-Scramble,

see Table 1 for sequences) were purchased from GenScript Corp, had greater than 95% purity, were

prepared by dissolution in milli-Q water to a concentration of 2 mmol/L and stored at -20°C. Peptides are

stable for one year following synthesis and were used within this time period.

9

Peptide Name Amino Acid Sequence

TAT-CBS-His

TAT-CBS

TAT-NC-His

TAT-NC

TAT-Scramble

RRRQRRKKRGGGAEFSARSRKRKANVTVFLQDHHHHHH

RRRQRRKKRGGGAEFSARSRKRKANVTVFLQD

RRRQRRKKRGVDIDQARLKMLGQTRPHDDDDCHHHHHH

RRRQRRKKRGVDIDQARLKMLGQTRPHDDDDC

RRRQRRKKRGFAFGRQVNKARSEKALGVSDRT

Table 1. Amino acid sequences of synthetic peptides used.

Table 1

10

2.7.3 TAT-mediated peptide delivery

For peptide treatment, cells were seeded in 24-well plates, grown to 60% confluence, washed

twice with PBS and administered solutions containing varying doses of peptide and their appropriate

serum-free medium (Medium 231 without SMGS for human aortic SMC, Medium 200 without LSGS for

human aortic EC, DMEM without FBS for MEF) for 1 h at 37°C. Cells were then washed twice with

PBS, and immediately given complete medium (Medium 231 containing SMGS, Medium 200 containing

LSGS or DMEM supplemented with FBS).

2.7.4 Cell counting

Primary human aortic SMC were seeded in a 96-well plate and treated with 100 µmol/L TAT-

CBS, TAT-NC or TAT-Scramble. 72 h post-treatment, cells were trypsinized, and counted with a

haemocytometer. Each experiment was done in triplicate.

2.7.5 Tritiated-thymidine incorporation assay

Incorporation of 3H-thymidine during DNA synthesis was used as a marker of cell proliferation.

After treatment with peptides, 0.5 µCi of 3H-thymidine (NET027Z, Perkin-Elmer, Waltham, MA) was

administered to each well of a 24-well plate. Cells were grown at 37°C and analyzed at 24, 48 and 72 h

after 3H-thymidine exposure. At the time of harvest, cells were washed twice with ice-cold PBS, and

incubated with 10% tricholoroacetic acid (TCA) on ice for 10 min to precipitate macromolecules. Two

more incubations with fresh 10% TCA on ice were carried out for 5 min each. TCA was removed, and

precipitates were dissolved by vigorous shaking at room temperature for 5 min with a solution of 0.2

mol/L NaOH and 1% SDS. Solubilized contents of wells were removed and added to 2.5 ml of

scintillation fluid (ReadySafe™ Cocktail, 141349, Beckman Coulter, Fullerton, CA). Radioactivity was

measured in a scintillation counter (LS 6500, Beckman Coulter). Measured counts were taken as relative

indices of proliferation by normalizing to counts from untreated cell controls.

2.7.6 LDH assay

The LDH-based in vitro toxicology assay kit (Sigma) was used to assess cytotoxicity of TAT-

peptide-treated cells. Human aortic SMC were plated 2 x 104 cells per well on a 24-well plate with phenol

red-free DMEM and treated with peptide. After 24 and 72 h, 500 µl of cell culture media was collected

from each well, followed by centrifugation (12,000 rpm, 30 min, 4oC) to completely remove debris.

Subsequent enzymatic assay was performed as per the manufacturer’s protocol.

11

2.7.7 Caspase-3 assay

The caspase-3 Colorimetric Assay Kit (Sigma, CASP3C-1KT) was used to assess apoptotic

activity of TAT-peptide-treated cells. Human aortic SMC were plated 2 x 104 cells per well on a 24-well

plate. Cell lysates were collected 24 and 72 h post-treatment for caspase-3 activity. Subsequent

enzymatic assay was performed as per the manufacturer’s protocol.

2.7.8 TUNEL staining assay

The In Situ Cell Death Detection Kit (Roche, 11684817910) was used to assess in situ apoptotic

activity of TAT-peptide treated cells. Human aortic SMC were seeded onto sterile coverslips in a 6-well

plate. Cells were fixed 72 h post-treatment and TUNEL staining performed as per the manufacturer’s

protocol.

2.7.9 Mouse carotid artery injury

All animal experimentation was conducted in accordance with operating protocols approved by

the Toronto General Hospital Animal Care Committee. 8-12 weeks age (20-25 g body weight) C57bl6

male mice were purchased from the Charles River Co. (Wilmington, MA) and housed for 1-2 wks before

experimentation. Animals were anesthetized using intraperitoneal ketamine-HCl (100 mg/kg IP) xylazine-

HCl (10 mg/kg, IP), and placed on a warming pad to regulate temperature. The primary bifurcation of the

right common carotid artery was isolated after midline neck incision, and two ligatures were placed

around the external branch. Next, the distal ligature was tied, and flow through the common carotid artery

was temporarily occluded with a vascular clamp. An incision was made in the external carotid artery

between the two ligatures, and a curved polished copper wire (0.3 mm diameter) introduced into the

lumen. The wire was advanced past the primary bifurcation into the common carotid artery, and vessel

systematically injured by simultaneously rotating the curved copper wire while passing along the vessel

four times. The wire was removed, and the external carotid artery tied off proximal to the incision with the

second ligature. The vascular clamp was next removed restoring flow through the common carotid artery.

The skin was closed with a single suture, and animals were allowed to recover on a warming pad.

2.7.10 Pluronic gel administration of TAT-CBS

Pluronic F-127 gel (BASF Corp, Mount Olive, NJ) was made up to 25% wt/vol and stored at 4˚C.

Immediately before use, peptides were added to pluronic gel to create 250 μmol/L solutions and kept on

ice. Post-carotid artery injury, the injured left common carotid artery was surrounded with 100 µl pluronic

gel with peptide with a syringe prior to wound closure.

12

2.7.11 BrdU administration

BrdU (Invitrogen, B23151) was made up to 6.25 mg/ml in PBS and stored at -20˚C. Mice were

weighed and given IP injections of BrdU (50 mg/kg) 17, 9 and 1 h before carotid artery harvest. Average

weight per mouse was around 20-25 g, and average volume of injection was around 200 μl.

2.7.12 Tissue processing and histology

For morphometry and collagen analysis, animals were lethally anaesthetized with pentobarbital

(100 mg/kg). Mice were perfusion-fixed at physiological pressure with 4% buffered paraformaldehyde

via LV puncture. Left and right common carotid arteries were harvested, fixed in 10% formalin in PBS,

embedded in paraffin and cut into 8 µm sections. Paraffin-embedded sections of control (TAT-NC-

treated) arteries were stained for H and E to determine the extent of injury formation. Prior to examining

TAT-CBS-treated arterial sections, three distances from the carotid artery bifurcation were identified as

points of maximal injury: Point A=200 µm, B=700 µm, C=1600 µm. Paraffin-embedded sections from

these 3 points of maximal injury were stained for Mason’s Trichrome and Picro-Sirius Red. For

assessment of re-endothelialization, arteries were harvested 7 days post-injury and paraffin-embedded. 4

μm sections were used 1 mm below the bifurcation of the common carotid artery. For His-tagged peptide

immunostaining, a mouse was sacrificed 4 days post-injury and the arteries were paraffin-embedded. 4

μm sections were used 200 μm below the bifurcation of the common carotid artery. For assessment of in

vivo SMC phenotype and proliferation, fresh carotid arteries were harvested 14 days post-injury, rinsed

in PBS embedded in OCT and snap-frozen by submerging cryomolds in a container of 95% ethanol (kept

at -80˚C overnight prior to use) floating in liquid nitrogen over dry ice. Frozen blocks were cut into 5 µm

sections within the area of maximal injury: 200 µm, 450 µm and 700 µm below the bifurcation of the

common carotid.

2.7.13 Morphometry analysis

Images of Mason’s trichrome-stained slides were captured using a slide-scanner (Olympus,

BX6IVS) and corresponding OlyVIA software. Images were analyzed with Adobe Photoshop CS4

software. Using the Lasso tool, the outlines of the external elastic lamina (EEL), internal elastic lamina

(IEL) and lumen were traced and the areas recorded. Cross-sectional thickness was calculated as follows:

(Media = EEL-IEL) and (Intima = IEL-Lumen).

2.7.14 Collagen analysis

Picro-Sirius Red-stained slides were examined with a polarized light microscope (Nikon Eclipse

Ti-S) and images captured with NIS-Elements software (Nikon). Polarized light microscopy images were

13

saved in RGB format. The red channel was extracted as type I Collagen and the green channel as type III.

Images were analyzed using Adobe Photoshop CS4 software. Area was measured using the Select Colour

Range and Record Measurements tools. Amount of collagen was recorded as percent total tissue by

normalizing to area of the bright field image.

2.7.15 Immunohistochemistry staining

TAT domain-mediated delivery of His-tagged peptides into human aortic SMC, EC and mouse

carotid artery was confirmed by immunostaining using rabbit anti-His antibody (sc-804, Santa Cruz). To

assess re-endothelialization, carotid artery sections harvested 7 days post wire-denudation injury were

immunostained with goat anti-CD31 antibody (sc-1506, Santa Cruz). Antigen-antibody complexes were

visualized by using rabbit or goat Vectastain kits (PK6101, PK6105, Vector Laboratories). Slides were

examined with a light microscope (Nikon Eclipse Ti-S) and images captured with NIS-Elements software

(Nikon).

2.7.16 Immunofluorescent staining

To examine SMC contractile phenotype in TAT-CBS-treated human aortic SMC or injured

carotid arteries, SMC marker immunostaining using rabbit anti-SM22-α (ProteinTech, 10493-1-AP),

mouse anti-sm-MHC (Abcam, ab683) and mouse anti-α-SMA (Sigma, A2547), donkey anti-rabbit-Cy3

(Jackson Immunoresearch, 711-165-152) or goat anti-mouse-Cy2 (Jackson Immunoresearch, 115-225-

146) antibodies. Cells were seeded on sterile coverslips in 6-well plates and treated with 100 µmol/L

TAT-CBS or TAT-NC. Untreated controls were cultured in complete media or serum-starved. Cells were

immunostained 72 h post-treatment. Cells and frozen sections were fixed with Cytofix (BD, 554655) for

10 min, washed with PBS 3 times for 5 min each, and permeabilized with BD Perm/Wash (BD, 554723)

for 20 min. Antibody incubations were performed with Dako Antibody Diluent (Dako, S3022-81). All

cells and frozen sections were incubated with primary antibody overnight at 4˚C, washed with PBS 3

times, incubated in secondary antibody + Hoechst nuclear stain (1:200 dilution, 1 mg/ml) for 1 h at RT,

and washed 3 times with PBS. To examine in vivo SMC proliferation of TAT-CBS-treated injured

carotid arteries, frozen sections were immunostained with the same protocol using mouse anti-BrdU-

Alexafluor 680-conjugated antibody (Invitrogen, A31859). After fixation and permeabilization, DNA was

denatured by incubation with 1N HCl (in H2O) for 30 min at room temperature prior to blocking with 5%

goat serum in 0.5% BSA in PBS for 15 min. After primary antibody incubation, sections were blocked

again before incubation with Hoescht. Mounted slides were examined with a confocal microscope

(Olympus Fluoview 1000) and images captured with FV10-ASW software.

14


One-way ANOVA was followed by post-hoc Bonferroni’s test. Data shown are mean + SE. IC50

calculations were determined by non-linear regression curve-fitting to an inhibitory dose-response curve.

Analyses were performed on Graphpad Prism v5.0 (GraphPad Software Inc, La Jolla, CA).

2.8 Results

2.8.1 Modification of the CBS peptide to increase bioavailability

As peptide nucleofection is not possible in vivo, we fused the TAT protein transduction domain

from HIV-1 to CBS to enable potential in vivo therapy (Fig. 1A). This approach was based on reports of

the TAT domain’s ability to successfully deliver a size-independent variety of molecules into cell nuclei.

While the exact mechanism of TAT-mediated protein transduction is unknown, it is believed that the

large cationic charge of the arginine residues allows TAT-conjugated proteins to effectively cross the cell

membrane by receptor-mediated endocytosis61, 62. Cells were exposed to TAT-CBS-His peptide in serum-

free cell culture media for 1 h, as serum decreases the transfection efficiency of the TAT domain61.

Delivery of TAT-CBS-His was confirmed by immunostaining using an anti-His tag antibody (Fig. 1B),

demonstrating that TAT-conjugation is an effective method of peptide delivery for TAT-CBS in HA-

SMC in vitro, and possibly in vivo.

2.8.2 TAT-CBS decreases the number of human aortic smooth muscle cells as measured by cell

counting

To further validate previous work with CBS in mouse aortic SMC and explore the clinical

relevance of the TAT-CBS peptide, we sought to determine whether the anti-proliferative effect of CBS is

evident in human aortic SMC (HA-SMC). Human aortic SMC were treated with TAT-CBS or one of two

negative control peptides: 1) TAT-NC: a random amino acid sequence of the same length as CBS, or 2)

TAT-Scramble: the scrambled CBS sequence, harbouring all of the original residues at different locations

(Table 1). Cell counting revealed that TAT-CBS significantly reduces the number of human aortic SMC

compared to untreated cells and compared to negative control peptide treatment (Fig. 2).

2.8.3 TAT-CBS decreases proliferation of human aortic smooth muscle cells as measured by 3H-

thymidine incorporation

To further establish that CBS works via an inhibitory effect on S-phase entry and to ascertain if

the effect was evident in human VSMC, we next examined DNA synthesis as measured by 3H-thymidine

incorporation in human aortic SMC treated with TAT-CBS over a range of concentrations (Fig. 3A).

15

ARRRQRRKKRGGGAEFSARSRKRKANVTVFLQDHHHHHH

TAT domain 6X His-tagCBS Sequence

BTAT-CBS-His TAT-CBS

Figure 1. TAT-CBS-His peptide delivery in vitro. (A) Sequence of TAT-CBS-His peptide. The 10 amino acid sequence of the TAT domain of HIV-1 was fused to the N-terminus of CBS, while a 6X His tag was fused to its C-terminus. (B) TAT domain-mediated delivery of CBS peptide into human aortic SMC. Human aortic SMC were treated with TAT-CBS-His (left panel) or TAT-CBS (right panel) peptides (100 μmol/L). Cells were fixed with 4% paraformaldehyde, and immunostained using rabbit anti-His antibody 72 h post-treatment. TAT-CBS has a transduction efficiency of approximately 100% in human aortic SMC.

Figure 1

16

TAT-CBS TAT-NC TAT-Scramble0.00

0.25

0.50

0.75

1.00

% P

rolif

erat

ion

(Cel

l Num

ber)

*

**

Figure 2. Anti-proliferative effect of TAT-CBS in human aortic SMC measured by cell counting. Primary human aortic SMC were treated with TAT-CBS, TAT-NC or TAT-Scramble (100 µmol/L). Proliferation was assayed by cell counting 72 h post-treatment. (Experiment repeated once, each condition performed with five replicates, *P<0.05, **P<0.01 by one-way ANOVA and post-hoc Bonferroni’s test).

Figure 2

17

BHuman Aortic EC

0.1 1 10 100 1000 10000-50

0

50

100

150

200

250TAT-CBSTAT-NC

Dose (µmol/L)

% P

rolif

erat

ion

([3 H

]TdR

Inco

rpor

atio

n)

*

A

0.1 1 10 100 1000 10000-50

0

50

100

150

200

250TAT-CBSTAT-NC

Dose (µmol/L)

% P

rolif

erat

ion

([3 H

]TdR

Inco

rpor

atio

n)

**

Human Aortic SMC

Figure 3. Anti-proliferative effect of TAT-CBS in human vascular cells as measured by 3H-thymidine incorporation. Dose-response curves in primary human aortic SMC (IC50=17.67 µmol/L) and EC (IC50=13.58 µmol/L) as measured by 3H-thymidine incorporation proliferation assays 72 h after peptide treatment. Measured counts were taken as relative indices of proliferation by normalizing to untreated cell controls. Non-linear regression fitting to an inhibitory dose-response curve was performed to generate IC50 values (each condition tested in triplicate; N=3 experiments, *P<0.05 for TAT-CBS vs. TAT-NC by one-way ANOVA and post-hoc Bonferroni’s test).

Figure 3

18

Results showed that compared to untreated cells, TAT-CBS, but not TAT-NC, was able to produce a

dose-dependent inhibitory effect on S-phase entry in human aortic SMC (IC50=17.67 μmol/L)

2.8.4 TAT-CBS decreases proliferation of human aortic endothelial cells

To explore the potential of TAT-CBS as a novel therapeutic agent, the effect of TAT-CBS on 3H-

thymidine incorporation in human aortic EC (HA-EC) was also examined (Fig. 3B). Results showed that

while compared to untreated cells, TAT-CBS did appear to cause a dose-dependent inhibition of

proliferation in human aortic EC (IC50=13.58 μmol/L), the effect was only significantly different from the

TAT-NC-treated cells at the highest dose of peptide administered (1 mmol/L), which may be cytotoxic63.

Interestingly, the 100 µmol/L dose of TAT-CBS significantly decreased proliferation compared to TAT-

NC treatment in human aortic SMC, but not EC. This difference may be insignificant, or could illustrate a

small degree of differential susceptibility of TAT-CBS between the two cell types.

2.8.5 Anti-proliferative effect of TAT-CBS is dependent on cyclin E

To confirm the mechanism of TAT-CBS cell cycle-inhibitory action occurs specifically through

interaction with cyclin-E, cyclin E1/E2 double knock-out mouse embryonic fibroblasts (MEF) were

obtained and treated with TAT-CBS. Consistent with the results from previous nucleofection experiments

with CBS (Appendix 1) the ability of TAT-CBS to block S-phase entry as measured by 3H-thymidine

incorporation was only evident in wild-type MEF (Fig. 4A), and not cyclin E1-/-E2-/- MEF (Fig. 4B).

These data show a cyclin E-dependent effect of the CBS peptide sequence on S-phase progression, which

supports the notion that TAT-CBS functions by mimicking the CaM-binding site on Cyclin E, and the

value of targeting this mechanism for developing anti-proliferative agents.

2.8.6 TAT-CBS does not increase cytotoxicity in human aortic smooth muscle cells

It is possible that observed anti-proliferative effects may be due to TAT-CBS-induced

cytotoxcity, not quiescence. To confirm that the anti-proliferative effect of TAT-CBS is truly due to cell

cycle arrest and not cell death, TAT-CBS-treated human aortic SMC were assayed for LDH release,

caspase-3 activation and TUNEL staining as indices of necrosis and apoptosis (Fig. 5). Results revealed

that the effective anti-proliferative dose of TAT-CBS (100 µmol/L) did not increase extracellular LDH

release, caspase-3 activation or TUNEL staining in human aortic SMC at 24 h (LDH and caspase-3, data

not shown) or 72 h post-treatment compared to untreated or negative control peptide-treated cells,

verifying TAT-CBS produces a decrease in proliferation through cell cycle arrest. The TAT-CBS

peptide’s lack of cytotoxicity in human VSMC supports its potential for possible clinical application.

19

A

B

0

100

200

300

% P

rolif

erat

ion

([3 H

]TdR

Inco

rpor

atio

n)

TAT-CBS TAT-NC TAT-CBS TAT-NC 10 µmol/L 100 µmol/L

Cyc E1/E2 KO MEF

0102030405060708090

% P

rolif

erat

ion

([3 H

]TdR

Inco

rpor

atio

n)

WT MEF

TAT-CBS TAT-NC TAT-CBS TAT-NC 10 µmol/L 100 µmol/L

**

Figure 4. Cyclin E–dependent anti-proliferative effects of TAT-CBS measured by 3H-thymidine incorporation. (A) MEF treated with TAT-CBS or TAT-NC peptides. Proliferation was assayed by 3H-thymidine incorporation 48 h post-treatment. Wild-type MEF demonstrate an anti-proliferative effect of TAT-CBS vs. TAT-NC (experiment repeated twice, N=6, **P<0.01 by one-way ANOVA and post-hoc Bonferroni’stest). (B) In Cyclin E1/E2 double knockout (DKO) MEF, TAT-CBS did not inhibit proliferation (experiment repeated once, n=3; P=NS by one-way ANOVA).

Figure 4

20

0

0.05

0.1

0.15

0.2

0.25

0.3

10 100 10 100 10 100 0 µmol/L peptide

TAT-CBS TAT-NC TAT-Scramble

OD

490

LDH Release Assay

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

10 100 10 100 10 100 0 µmol/L peptide

TAT-CBS TAT-NC TAT-Scramble

OD

405

Caspase-3 Activity Assay

A

B

Figure 5

21

Human Aortic SMC

0 100 200 300 40002468

1040

50

60

70PBS (100 µM volume)TAT-CBS (100 µM)TAT-NC (100 µM)PBS (10 µM volume)TAT-CBS (10 µM)TAT-NC (10 µM)(-) Control(+) Control

DAPI+

TUN

EL+

DAPI TUNEL

(+) Control(DNase)

100 µMTAT-CBS

C

Figure 5. TAT-CBS does not increase cell death in human aortic SMC. (A) LDH release, (B) caspase-3 activity and (C) TUNEL staining assays were performed 72 h following peptide treatments. TAT-CBS treatment did not increase LDH release or caspase-3 activity compared to TAT-NC or untreated controls. Similar results were obtained at 24 h (not shown) (n=4 for both studies, P=NS by one-way ANOVA). TAT-CBS treatment causes a dose-dependent reduction in cell number, but does not increase the amount of apoptosis measured by TUNEL staining compared to controls. Representative fluorescent images are shown.

Figure 5

22

2.8.7 TAT-CBS does not alter differentiation of human aortic smooth muscle cells as measured by

contractile smooth muscle cell marker immunofluorescent staining

In order to further understand mechanisms of TAT-CBS action, we sought to examine if peptide

treatment prevented proliferation and remodeling by altering differentiation of VSMC. Therefore, TAT-

CBS-treated human aortic SMC were immunostained for contractile SMC marker proteins smooth muscle

22-α (SM22-α), smooth muscle myosin heavy chain (sm-MHC) and α-smooth muscle actin (αSMA) (Fig.

6). Compared to untreated, quiescent (serum-starved) and TAT-NC-treated controls, TAT-CBS-treated

human aortic SMC did not appear appreciably differentiated, “de-differentiated” or synthetic, or

transformed. Therefore, TAT-CBS treatment does not appear to significantly alter the differentiation

status of human aortic SMC in vitro, attesting to the peptide’s ability to cause a cell cycle-specific effect

without increasing de-differentiation.

2.8.8 TAT-CBS appears to have greater transduction efficiency in human aortic smooth muscle vs.

endothelial cells as indicated by His-tag immunostaining

As an indicator of TAT-CBS peptide transduction efficiency and intracellular lifespan, human

aortic SMC and EC were treated with His-tag-conjugated TAT-CBS. His-tag immumostaining at 1, 4, 8,

24 and 72 h post-treatment revealed decreasing His immunostaining signal over time compared to un-His-

tag-conjugated peptide (Fig. 7). The CBS peptide appeared to have a lifespan of approximately 72 h in

vitro. Moreover, there appeared to be greater His-tag signal intensity in transfected HA-SMC compared to

HA-EC. Therefore, TAT-CBS appears to have increased transduction efficiency in HA-SMC compared to

HA-EC as indicated by His immunostaining in vitro. This may partially account for the difference in anti-

proliferative effect of the 100 µmol/L dose of TAT-CBS between the two cell types.

2.8.9 Pluronic gel administration of TAT-CBS in vivo causes peptide delivery into smooth muscle cells

of the arterial wall post-carotid injury

The TAT-CBS-His peptide (250 μmol/L) was previously tested in a mouse common carotid

artery injury model using pluronic gel administration. In aqueous solution, the surfactant pluronic F-127

transforms from a liquid to a non-fluid hydrogel at or above room temperature. This property of pluronic

F-127 is especially desirable for in vivo peptide delivery, as the semisolid solution of peptide and

surfactant allows the peptide to remain concentrated and protected by the surfactant matrix. Morphometry

previously showed that injured arteries treated with TAT-CBS-His reduced VSMC proliferation

compared to TAT-NC-His and vehicle controls (data not shown).

23

Serum + PBS

Serum-starve +PBS

Serum + TAT-CBS

Serum + TAT-NC

IgG

SM22-α sm-MHC α-SMA

Figure 6. TAT-CBS did not appreciably alter expression of smooth muscle cell markers in human aortic SMC. Human aortic SMC were treated with TAT-CBS or TAT-NC (100 µmol/L). Untreated (PBS) controls were serum-starved or exposed to serum-supplemented media. Cells were immunostained for SMC markers SM22-α, sm-MHC and α-SMA 72 h post-treatment. Representative confocal microscopy images are shown.

Figure 6

24

TAT- CBS TAT-CBS-His

1 h

TAT-CBS TAT-CBS-His

4 h

8 h

24 h

72 h

Human Aortic SMC Human Aortic EC

Figure 7. TAT-CBS appears to have greater transduction efficiency in human aortic SMC than EC. Human aortic SMC and EC were treated with either TAT-CBS-His or TAT-CBS (100 µmol/L ). Cells were immunostained 1, 4, 8, 24 and 72 h post-treatment with rabbit anti-His antibody. Representative images are shown.

Figure 7

25

To establish if this effect was indeed due to the anti-proliferative effect of CBS peptide, in vivo delivery

of TAT-CBS-His into VSMC of the mouse carotid artery was evaluated by immunostaining with an anti-

His antibody (Fig. 8). In vivo His-tag detection appeared heterogeneous. In the example shown, TAT-

CBS-His is only present in SMC of one side of the artery, whereas on the other side, TAT-CBS-His is

visible in the adventitia. Compared to peptide transduction in vitro, peptide transduction efficiency may

be decreased in vivo. This could be due to the method of peptide delivery, as adventitial His detection is

consistent with pluronic gel TAT-CBS-His administration to the adventitial surface of artery. Moreover,

this artery was harvested 4 days post-injury, as we predicted the in vivo lifespan of TAT-CBS to be less

than 14 days (the time point used to assess intimal-medial thickness). It is possible that at this early time

point, not all of the peptide has penetrated from the adventitia to the media, producing the anti-

proliferative effects seen at 14 days post-injury. Importantly, in the area of the artery where TAT-CBS-

His has entered the media, it appears to be exerting its expected anti-proliferative effect, as there is

reduced arterial wall thickening compared to other areas (Fig. 8 arrows).

2.8.10 Pluronic gel administration of TAT-CBS in vivo decreases thickness of the intima

As migration of VSMC from media to intima following initial proliferation in media is an

essential step in the development of restenosis or atherosclerosis, quantitative analysis of arterial sections

examining three parameters: (i) total area of the arterial media, (ii) total area of the arterial intima and (iii)

ratio of intima to media (I/M ratio) was previously performed on injured TAT-CBS-treated carotid

arteries 14 days post-injury.

In order to validate previous data and further investigate the in vivo mechanism of TAT-CBS,

morphometry was repeated on new sections in a separate, more rigourous analysis. As opposed to

randomly sampling nine sections per artery at predetermined, evenly-spaced distances from the

bifurcation, as per the previous analysis, morphometric examination was restricted to areas of maximum

carotid artery injury. To characterize appropriate areas, TAT-NC-treated arteries were sectioned in their

entirety and stained for H and E. Blinded to TAT-CBS-treated arteries, three distances from the common

carotid artery bifurcation were identified as points of maximum injury in TAT-NC controls (Point A=200

µm, B=700 µm, C=1600 µm). TAT-CBS and TAT-NC sections from these three points were then stained

for Mason’s trichrome and intimal-medial thickness compared (Fig. 9).

Confirming previous results, a significant reduction was observed in neointima formation and I:M

ratio in carotid arteries treated with TAT-CBS over TAT-NC. Moreover, the anti-proliferative effect

appeared graded from the carotid bifurcation to the distal carotid injury site. With this method of analysis,

we did not observe a significant anti-proliferative effect of TAT-CBS treatment compared to TAT-NC-

26

IgG control Anti-His antibody

Figure 8. TAT-CBS-His peptide delivery to SMC of an injured carotid artery. Arteries were harvested 4 days post-injury. Sections were immunostained using rabbit anti-His antibody (right panel) or rabbit IgG (left panel).

Figure 8

27

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31

Figure 9. TAT-CBS decreases neointimal formation post-carotid injury. (A) Schematic of carotid injury morphometric analysis. Mice were treated with peptides (250 μmol/L) in pluronic gel immediately after carotid injury. TAT-NC-treated injured carotid arteries were paraffin-embedded, cut into 8 µm sections and stained for H and E to determine the extent of injury formation. Prior to examining TAT-CBS-treated arterial sections, three landmark distances from the carotid artery bifurcation were selected as points of maximal injury: Point A, B and C. (B) Change in intima media thickness over distance. Average intima media quantification at points A, B and C are shown in relation to one another over distance. The effect of TAT-CBS treatment increases with distance from the bifurcation. (C) Intima media thickness at Point A, (D) B and (E) C. Representative images and average values are shown. TAT-CBS (n=6), TAT-NC (n=6), uninjured TAT-CBS (n=6) and uninjured TAT-NC (n=7). (P=NS for panels C and D *P<0.05 for panel E by one-way ANOVA and post-hoc Bonferroni’s test).

Figure 9

32

His at Point A, which was closest to the bifurcation. However, at Point B, there was an average trend of

decreased intimal and medial thickness in the TAT-CBS group. At Point C, morphometry showed

significantly decreased neointimal thickness and I:M ratio. Therefore, the putative anti-proliferative effect

of TAT-CBS on carotid injury appears to increase with distance from the carotid artery bifurcation. This

is likely a consequence of anatomy and the penetration of peptide using pluronic gel through the outer

surface of the artery, as opposed to the TAT-CBS peptide’s mechanism of action. Spatially-differential

degree of injury or peptide transfection may be inherent to wire carotid injury or pluronic gel peptide

delivery in our hands.

2.8.11 Pluronic gel administration of TAT-CBS in vivo decreases increases expression of smooth muscle

22-α

Proliferating cell nuclear antigen (PCNA) staining was previously employed as a measure of the

level of proliferation in injured arteries. Arteries treated with TAT-CBS-His had a decreased percentage

of PCNA-positive nuclei, confirming the ability of TAT-CBS-His peptide to inhibit cell proliferation in

injured arteries (Appendix 2). BrdU staining was performed as an additional index of proliferation in

injured arteries. Based on similar studies in mice post-carotid injury64, 65, 50 mg/kg BrdU was

administered via IP injection 17, 9 and 1 h before carotid artery harvest. Despite optimizing conditions in

positive controls, immunofluorescent staining did not reveal substantial BrdU signal compared to

negative control tissue (small intestine crypts of villi) in injured arteries (Appendix 3). This may be

because the time point of interest for measuring intima/media thickness is not the optimal time point for

observing SMC proliferation by BrdU incorporation. At 14 days post-injury, substantial SMC

proliferation may not be occurring within the 17 h window in which arteries were exposed to BrdU before

being harvested. Therefore, BrdU administration and tissue harvesting may need to be performed earlier

than 14 days post-injury (i.e. 7 days post-injury) with our model of wire carotid injury in order to observe

meaningful BrdU incorporation into SMC.

However, in vivo SM22-α immunostaining revealed that compared to TAT-NC injured arteries,

TAT-CBS-treated arteries have significantly increased SM22-α expression (Fig. 10). Injured TAT-NC-

treated arteries displayed decreased SM22-α expression compared to uninjured controls, which may be

indicative of SMC de-differentiation. Conversely, TAT-CBS-treated arteries possessed SM22-α

expression similar to uninjured arteries. Interestingly, it was recently shown that increased expression of

SM22-α can inhibit SMC proliferation and neointimal formation in carotid injury66. As SM22-α is a

contractile SMC marker, CBS-induced growth-arrest may prevent VSMC from de-differentiating in vivo.

33

NC CBS Uninjured

NC CBS0

1.0×10 6

2.0×10 6

3.0×10 6

4.0×10 6

5.0×10 6

***SM22

- αIn

tegr

ated

Den

sity

Figure 10. TAT-CBS treatment maintains SM22-α expression post-carotid injury. TAT-CBS or TAT-NC-treated carotid arteries were embedded in OCT , cut into 5 µm sections and immunostained for SM22- α (Cy3) and Hoescht nuclear stain. Representative confocal microscopy images are shown. SM22- α expression was quantified by measuring Integrated Density of the Cy3 channel in Adobe Photoshop CS4. Average values are shown (n=7) (*P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA and post-hoc Bonferroni’s test).

Figure 10

34

2.8.12 Pluronic gel administration of TAT-CBS does not affect collagen deposition in vivo post- carotid

injury

As extracellular matrix remodeling also contributes to the development of restenosis, the effect of

TAT-CBS treatment on collagen deposition was investigated. Collagen quantification was performed by

Picro-Sirius Red staining and subsequent polarized light microscopy in injured carotid arteries treated

with TAT-CBS 14 days post-injury (Fig. 11). Results show TAT-CBS did not affect accumulation of total

collagen, Collagen I or Collagen III fibres compared to TAT-NC-treated controls. Consistent with the

expected mechanism of TAT-CBS action, the data to date indicate that peptide treatment affects VSMC

proliferation and not extracellular matrix production.

2.8.13 Pluronic gel administration of TAT-CBS does not affect re-endothelialization in vivo post-carotid

injury

Given in vitro human aortic EC data, the effect of CBS on the re-endothelialization of injured

carotid arteries was also investigated in vivo. Interestingly, CD31 staining of sectioned arteries revealed

that TAT-CBS-His peptide delivery did not affect re-endothelialization at 7-days post-injury, as compared

to gel-only and TAT-NC-His-administered arteries (Fig. 12).

2.9 Discussion

2.9.1 Summary

2.9.1.1 Previous findings with CBS peptide

It has been previously shown that CBS, a specific 22 amino acid peptide: inhibits (i) the binding

of CaM to cyclin E, (ii) Ca2+-sensitive cyclin E/CDK2 activity (Appendix 4), (iii) G1 to S cell cycle

progression of VSMC, (iv) the activating phosphorylation of CDK2 at Thr160 without altering the

inhibitory phosphorylation on Thr14/Tyr15 by selectively interfering with CaM-cyclin E interactions (data

not shown). Importantly, (v) the binding of CaM to another target protein, calcineurin, was not altered by

CBS peptide (data not shown). Therefore, the CBS peptide is designed to selectively inhibit CaM-cyclin

E interactions, and does not appear to interfere with other Ca2+/CaM-dependent pathways.

2.9.1.2 TAT-CBS findings

In this study, we have shown that TAT-conjugated CBS peptide inhibits (vi) VSMC and EC

proliferation in vitro, (vii) appears to have increased transduction efficiency in VSMC vs. EC, (viii)

without increasing cytotoxicity, apoptosis or (ix) de-differentiation, and (x) did not inhibit proliferation of

cyclin E1/E2-deficient MEF. In a mouse model of carotid artery injury, in vivo delivery of the TAT-CBS-

His peptide to VSMC demonstrated (xi) decreased neointima thickness and (xii) increased smooth muscle

35

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Figure 11

36

Figure 11. TAT-CBS does not affect collagen deposition post-carotid injury. (A) Polarized light microscopy of Picro-Sirius Red-stained injured arteries. TAT-NC or TAT-CBS paraffin-embedded carotid arteries sections were stained for Picro-Sirius Red at the same three landmark distances (Point A = 200 µm, B = 700 µm and C = 1600 µm) from the carotid artery bifurcation of maximal injury determined from H and E staining. Representative images from point B are shown. (B) Collagen quantification over distance. Average collagen quantification at points A, B and C are shown in relation to one another. TAT-CBS treatment did not affect collagen deposition compared to TAT-NC-treated or uninjured controls at all three points of maximal injury. (C) Average collagen quantification by artery. Measurements at A, B and C were combined to created an average value over distance for each artery. TAT-CBS (n=6), TAT-NC (n=6), uninjured TAT-CBS (n=6) and uninjured TAT-NC (n=7), (P=NS by one-way ANOVA).

Collagen


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37

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Figure 12. TAT-CBS-His does not affect re-endothelialization in vivo. Sections of injured carotid arteries harvested 7 days post-injury were immunostained for CD31. Representative CD31-stained sections (10X objective) are shown. Percent re-endothelialization was quantified for F-127 (n=5), TAT-CBS (n=6), and TAT-NC (n=6) treated mice (P=NS by one-way ANOVA).

Figure 12

38

contractile protein expression, (xiii) without adversely affecting collagen deposition or (xiv) re-

endothelialization. Taken together, these findings suggest that the CBS peptide inhibits (a) cyclin E-

specific, Ca2+/CaM-dependent, CDK2 activity and (b) Ca2+-sensitive cell cycle progression and cell

proliferation in VSMC.

2.9.2 Implications

2.9.2.1 Selectivity for calcium-sensitive, rapid, pathological proliferation

These findings demonstrate that TAT-CBS has distinct characteristics that may set it apart from

current cell cycle inhibitors used to impede smooth muscle proliferation in vascular diseases. Firstly, CBS

has increased selectivity: it has been shown that CBS peptide does not affect basal levels of CDK2

activity; it selectively inhibits Ca2+-sensitive enhancement of CDK2 activity at the G1 to S cell cycle

phase transition, a feature that should restrict its effects to rapidly proliferating cells (Appendix 4). In

vascular diseases such as atherosclerosis and restenosis, SMC are proliferating in a pathological fashion

that is distinct from the low levels of regulated proliferation. Therefore, it is possible the peptide may

possess in vivo selectivity for pathological, unregulated, rapidly proliferating SMC in the wall of a

diseased artery over the endothelial lining.

2.9.2.2 Selectivity for smooth muscle vs. endothelial cell transduction

Moreover, although CBS possesses similar anti-proliferative potency in SMC and EC, it has

appears to have inherent specificity for delivery to SMC compared to EC both in vitro and in vivo.

Therefore, the TAT-CBS peptide may be less toxic to the endothelium, and represents a promising

potential novel therapy for vascular proliferative disorders.

2.9.3 Limitations

2.9.3.1 Potential non-specific TAT activity

The TAT transduction domain conjugated to the CBS peptide may possess inherent physiological

effects and exert non-specific effects on signalling pathways in VSMC. In fact, TAT-treatment has been

shown to reduce the vasodilatory capacity of porcine coronary arteries by decreasing endothelial nitric

oxide synthase production67. Therefore, TAT may similarly have non-specific effects on arterial function

in TAT-CBS-treated mouse carotid arteries. Although results showing the anti-proliferative effect of

TAT-CBS treatment is cyclin E-dependent indicate specificity of peptide action, the effect of the TAT

transduction domain alone on activation of various signalling cascades was not investigated in VSMC.

39

2.9.3.2 Pluronic gel delivery

Immunostaining revealed inconsistent transduction of TAT-CBS-His into SMC of the mouse

carotid artery via pluronic gel delivery. Although eccentric retention of TAT-CBS-His by mural VSMC

appeared to colocalize with prevention of neointima formation, these results highlight the importance of

delivery method when considering potential clinical application of TAT-CBS. Perhaps a more direct

delivery method to the inner surface of the artery such as via drug-eluting stent would improve uniformity

of in vivo peptide transduction efficiency.

2.9.3.3 Wire carotid artery injury

In vivo data show variable extent of injury along the length of the common carotid artery using

the current wire injury method. Additionally, the methodology used only examined one time point (14

days post-injury) for measurement of arterial wall thickness, and was not able to confirm increased

smooth muscle cell proliferation via BrdU incorporation at this time point. Other methods to confirm cell

cycle stage of VSMC were not performed post-injury. Therefore, this method of injury is possibly

inconsistent in our hands, and was not verified to increase smooth muscle cell proliferation except

indirectly by observations of increased arterial wall thickness compared to uninjured controls in our

studies.

2.9.3.4 Similar anti-proliferative effect in smooth muscle vs. endothelial cells

The main shortcoming of current smooth muscle cell cycle-inhibitory agents for proliferative

vascular diseases is non-specific, harmful effects on the endothelium. Unfortunately, results show that

TAT-CBS has similar anti-proliferative potency in SMC compared to EC in vitro. However, in vivo data

indicates that the method of peptide delivery may impact whether endothelial cells are affected by CBS.

Although TAT-CBS treatment did not affect re-endothelialization in our carotid injury model, this may be

because TAT-CBS was administered in pluronic gel to the outer surface of the artery, and was not able to

transfect across all of the cell layers to the inner endothelial lining. Therefore, regardless of the peptide’s

susceptibility for VSMC vs. EC, a method of peptide delivery exclusive to VSMC and not EC could be

envisioned for the CBS peptide.


2.9.4.1 Cell cycle analysis with TAT-CBS

It may be of interest to employ flow cytometry analysis to examine the cell cycle stage of TAT-

CBS-treated VSMC. Moreover, peptide-induced G1 cell cycle arrest could be confirmed through

40

investigation of cyclin D/cdk4 kinase activity or retinoblastoma phosphorylation using specific kinase

assays or Western blot analysis at multiple time points post-treatment both in vitro and in vivo.

2.9.4.2 CBS as a novel drug-eluting stent agent: smooth muscle cell-selective strategies

Based on our mouse carotid injury model data, we speculate that the CBS peptide could be

delivered to patients at the time of coronary intervention and continuously administered afterward as a

novel agent for DES. VSMC-targeted delivery of CBS peptide or its next generation surrogates may have

the potential to selectively block rapidly proliferating VSMC, while not interfering with reformation of

the anti-thrombotic endothelial cell lining at the percutaneous coronary intervention (PCI) site. For

instance, a gene therapy method could be employed in which a plasmid containing the CBS sequence is

under the transcriptional control of a smooth muscle-specific promoter. This approach has proven

effective with the SM22-α promoter for in vivo smooth muscle-specific transfection in a rat model of

carotid injury68. Additionally, virus retargeting techniques that modify surface proteins and moieties for

binding to a cell surface receptor specific to vascular smooth muscle cells may further enhance delivery69-

71. Our in vitro data also suggest that as a therapeutic peptide, CBS could have greater specificity and

decreased toxicity compared to current pharmacological agents.

2.9.4.3 Promise of small molecule-based therapies

Similar to CBS, other small molecules such as peptides are successfully being utilized as

inhibitors of protein-protein interaction and are currently being marketed or are in various stages of

development72. Recently, a peptide inhibitor of the nuclear factor of activated T-cells (NFAT) was

developed and observed to selectively inhibit NFAT-mediated proliferation and inflammation of

VSMC73. Small peptides that block the interaction of cyclin A/CDK2 with substrates such as E2F1 have

also been investigated in a number of tumour cell lines. These inhibitory peptides induced S phase arrest

and abrupt apoptosis. Cell death was selective to transformed cells; although a normal human fibroblast

cell line did not undergo apoptosis, a T antigen–transformed subclone derived from it was killed74.

Similar to the CBS peptide, other molecules have been developed to inhibit the binding of CaM to its

target proteins, such as ATPase75 and MLCK76. The success of these related drugs supports further drug

development based on CBS.

41

CHAPTER 3. NOVEL CALCIUM-SENSITIVE MECHANISMS OF VASCULAR SMOOTH

MUSCLE CELL CYCLE CONTROL

Sonya Hui, Mansoor Husain.

42

3.1 Abstract

Mechanisms regulating cell cycle progression in vascular smooth muscle cells (VSMC) are

potential therapeutic targets for atherosclerosis and restenosis. Having shown that G1 to S phase

transitions of VSMC are dependent on increases in cytosolic calcium (Ca2+) concentrations, we

hypothesized that the cell cycle inhibitor p27Kip1 (p27) was a putative mediator of this Ca2+-sensitivity,

and that increased [Ca2+] would reduce p27 levels via CaM-dependent phosphorylation of p27 (P-p27)

and subsequent proteasomal degradation. We sought also to identify novel Ca2+-sensitive proteins

involved in cell cycle control of VSMC.

Western blots of lysates from cell cycle-synchronized mouse VSMC (MOVAS) revealed that

p27 levels were notably reduced but still detectable, while P-p27 levels were increased, in G1-stage (4 h

after serum stimulation) as compared to G0-stage (serum-starved) MOVAS. In situ analyses were

performed on synchronized MOVAS between G0 and G1 using the membrane-permeable Ca2+-chelator

BAPTA-AM and the Ca2+-ionophore ionomycin prior to protein extraction and Western blot. G0-

synchronized MOVAS did not show Ca2+-dependent differences in p27 levels or phosphorylation. By

contrast, G1-stage cells showed that ionomycin-increased [Ca2+ ] caused significant decreases in p27

levels with reciprocal increases in P-p27 over time. As CaMK-II/MEK/ERK-mediated proteosomal p27

degradation occurs in human adenocarcinoma cells, inhibitors of CaMK-II, MEK and the proteasome

were tested in MOVAS. While MEK inhibition had no effect, inhibitors of CaMK-II and proteasome

prevented p27 degradation and phosphorylation during G1. To survey other potential Ca2+/CaM-

associated proteins involved, whole cell extracts from G0- and G1-stage MOVAS were incubated with

CaM-sepharose resin. Bound proteins were eluted, subject to SDS-PAGE and Coomassie staning. A band

that differed significantly between G0 and G1 samples was identified by mass spectroscopy as the CaM-

binding GTPase IQGAP1. Interestingly, Western blots from cell cycle-synchronized MOVAS revealed

uniform expression of IQGAP1 across cell cycle stages.

Ca2+-sensitive phosphorylation and degradation of p27 in VSMC is specific to the G1-stage of the

cell cycle and mediated by CaMK-II and the proteasome, but not MEK. With no change in total IQGAP1,

CaM-bound IQGAP1 is increased during G1 progression. These data identify two new potential

therapeutic targets amongst the Ca2+-sensitive cell cycle regulators of VSMC.

43

3.2 Introduction

3.2.1 Calcium-sensitive targets of cell cycle control

Intracellular calcium (Ca2+) transients are known to regulate cell cycle progression in a variety of

cell types, but the mechanisms by which this occurs are not well-characterized28. We recently showed that

in vascular smooth muscle cells (VSMC), the major calcium-transducer calmodulin (CaM) binds directly

to the cell cycle regulator Cyclin E, and is necessary for G1-to-S phase progression55. Cyclin E is one

member of a growing family of cell cycle regulators identified as targets of Ca2+/CaM signalling. For

instance, CaM is also necessary for cdk4 activity and cyclin D/cdk4 nuclear accumulation in rat kidney

cells35, and Calmodulin Kinase I (CaMK-I) regulates cyclinD1 migration and cdk4 activation in human

fibroblasts77. CaM also binds directly to the cell cycle inhibitor p21Cip1(p21) through a CaM-binding

domain at its carboxy-terminal36, and it is necessary for its nuclear localization78. These are just a few

examples of currently known calcium-sensitive cell cycle regulators; there are likely more that have yet to

be identified.

3.2.2 Putative mechanism of calcium-sensitive cell cycle control in vascular smooth muscle cells:

p27Kip1

Interestingly, preliminary data from our lab shows that increased levels of Ca2+/CaM may

accelerate the degradation of the cell cycle inhibitor p27Kip1 (p27) in VSMC. This could represent an

important undiscovered calcium-sensitive mechanism of vascular smooth muscle cell cycle regulation

similar to CaM-cyclin E interaction.

3.2.3 Cell cycle inhibitor p27Kip1

The p27 protein is a cyclin-dependent kinase inhibitor that is part of the larger Kinase Inhibitor

Protein (KIP) family that inhibits G1-associated cyclin/cdk complexes79. Overexpression of p27 leads to

G1 cell cycle arrest in human cells80, whereas p27 knock-out mice display a phenotype of gigantism,

multi-organ hyperplasia and tumorigenesis, demonstrating the requirement of p27 for growth control81, 82.

P27 is traditionally known as an inhibitor of cdk2, although it has also been shown to target cdk1 and

cdk483. P27 is considered part of a family of “intrinsically unstructured” proteins, which are capable of a

variety of possible conformations and binding to several different targets83. Therefore, defining a

straightforward general mechanism of p27 function has been complicated by seemingly inconsistent

findings.

44

3.2.4 Complexity of p27 regulation

3.2.4.1 Classic p27 degradation

In accordance with its function as a cell cycle inhibitor, p27 is highly expressed during

quiescence and markedly down-regulated during proliferation84. This primarily occurs through control of

p27 protein degradation and translation, as p27 mRNA levels remain constant across the cell cycle85. In

order to initiate cell cycle re-entry in quiescent cells, p27 is phosphorylated by cyclin E/cdk2 at its

threonine (Thr) 187 residue86. Phosphorylated p27 is subsequently recognized by the SCFSkp2 (Skp2)

ubiquitin ligase and degraded by the proteasome82, 87.

3.2.4.2 Non-classical mechanisms of p27 degradation

However, classic understanding of p27 degradation is complicated by findings that the ubiquitin

ligase Kpc can recognize and degrade unphosphorylated p27 during G188. Moreover, studies have shown

that during the S phase of the cell cycle, p27 can be cleaved into an inactive form through a ubiquitin-

independent mechanism89. During early G1, a caspase-cleaved form of p27 has also been detected in the

absence of apoptosis90. These findings suggest the existence of other putative mechanisms of p27

degradation in addition to classic Skp2 ubiquitin ligase proteasome. Furthermore, several tyrosine kinases

have been found to phosphorylate p27 such as Lyn, which acts on Tyr 88, Abl, which acts on Tyr 88 and

8979, and Src which acts on Tyr 74 and 8891. Collectively, tyrosine phosphorylation has been shown to

increase Thr 187 phosphorylation, decreasing p27 protein stability and enhancing degradation79.

Therefore, in addition to alternative degradation pathways outside of Skp2-mediated proteolysis, tyrosine

kinase phosphorylation and possibly other signalling pathways can modulate p27 degradation.

3.2.5 Calcium signalling and p27 regulation

The abundance of pathways surrounding p27 degradation support the plausibility of potential

Ca2+/CaM-signalling involvement in its regulation. Indeed, it was shown in Alzheimer’s disease

lymphoblasts that disease-associated p27 down-regulation can be abrogated by treatment with CaM

antagonists92. Moreover, Li et al. recently defined a calcium-sensitive mechanism of p27 degradation in

human colon adenocarcinoma cells, in which activated calmodulin-dependent kinase Calmodulin Kinase

II (CaMK-II) initiates classic Thr 187/Skp2 ubiquitin-mediated degradation through intermediate

MEK/ERK phosphorylation93.

Studies also show that p27 can be degraded by the calcium-sensitive proteases calpains during

adipocyte differentiation, and in a MAP Kinase-dependent manner in choroidal melanoma cells94, 95.

Interestingly, the opposite effect of calcium on p27 has also been demonstrated. In conjunction with a

decrease in c-myc expression, increased intracellular calcium can actually up-regulate levels of p27 in

murine B-lymphoma cells96. Inhibition of SMC proliferation and p27 upregulation can also be induced by

45

genetic knockout of calcium-sensitive phosphodiesterase (PDE) 1A97. Considering the complexity of

current evidence regarding calcium signalling and p27, there likely exists a myriad of calcium-sensitive

p27 regulatory mechanisms that are differentially manifested depending on cell type, cell cycle stage and

extracellular signal, among other factors95.

3.3 Rationale

3.3.1 Implication of p27 in inhibition of smooth muscle cell proliferation

Findings suggest that p27 is an important mediator of proliferation in VSMC. Firstly, the

existence of classic Skp2-mediated ubiquitin proteasomal p27 degradation in response to serum-

stimulation has been confirmed in VSMC98. Moreover, experiments show that inhibiting proliferation

through various signalling pathways such as peroxisome proliferator-activated receptor-delta (PPAR-

δ)/nitric oxide synthase (NOS) activation99, Nurr1 overexpression100, and serum response factor (SRF)

silencing101 all result in p27 upregulation in VSMC. Taken together, these results signify that p27 may be

part of a common final pathway of inhibited SMC proliferation. In addition to the activation of signalling

pathways, inhibition of SMC proliferation by treatment with anti-proliferative agents for restenosis such

as fluvastatin and everolimus102, rapamycin103 and heparin104 also produce increased p27 expression.

Therefore, related results from endogenous proliferating VSMC, specific signalling pathways in VSMC

and VSMC treated with anti-proliferative pharmacological agents indicate that p27 is a significant

controller of VSMC cycle progression.

3.3.2 Expression of p27 in proliferative vascular pathologies: restenosis and atherosclerosis

Furthermore, p27 is implicated in proliferative vascular pathologies such as in-stent restenosis

and atherosclerosis. For instance, a mutant form of p27 lacking a binding site for its target molecule cdk2

was unable to inhibit SMC migration and proliferation in vitro105. In vivo Skp2-mediated downregulation

of p27, increased SMC proliferation, and neointimal hyperplasia have been demonstrated in animal

models of carotid artery injury106, 107. Moreover, both partial and complete genetic inhibition of p27 in

hypercholesteremic mice caused acceleration of atherosclerosis105. Meanwhile, increased p27 expression

is observed in advanced human atherosclerotic plaques108 and in rat carotid angioplasty 2 weeks post-

injury109, indicating a role for p27 upregulation during later time points of atherosclerotic development

following initial injury-induced downregulation110. Therefore, the establishment of p27 calcium-

dependence may provide new insight into mechanisms of vascular disease, as well as form the basis for

development of effective novel treatments. Overall, investigation of p27 calcium-dependence and other

calcium-sensitive mechanisms of cell cycle regulation in VSMC is clinically significant as a therapeutic

strategy for proliferative vascular pathologies.

46

3.4 Objectives

3.4.1 Investigation of putative calcium/calmodulin-sensitive p27 degradation

We will clarify possible calcium-dependence of p27 expression by studying the effect of

increased calcium on p27 protein stability. This may identify p27 as a novel calcium-sensitive regulator of

cell cycle progression in VSMC, and as another target for future clinical development.

3.4.2 Novel calcium/calmodulin-sensitive mechanisms of cell cycle control

Moreover, we aim to identify other novel calcium/calmodulin-sensitive targets of cell cycle

control by surveying CaM-binding proteins in VSMC for cell cycle-dependent differential expression,

which may indicate involvement in both calcium signalling and cell cycle control.

3.5 Hypothesis

We hypothesize that increased intracellular Ca2+ in VSMC causes reductions in p27 protein levels

through CaM-dependent enhancement of Thr 187 phosphorylation and subsequent proteasomal

degradation.

3.6 Materials and Methods

3.6.1 Cell culture

Isolation and culture of primary aortic mouse SMC, and characterization of the immortalized

mouse SMC line MOVAS have been previous described58. MOVAS were cultured in DMEM with 10%

FBS (HyClone, SH30070.03) and 1% penicillin-streptomycin (Invitrogen, 15070-063). Wild-type and

cyclin E1/E2 double knockout (Cyc E DKO) mouse embryonic fibroblasts (MEF) were kindly provided

by Dr. P. Sicinski (Harvard Medical School), and maintained in DMEM with 10% FBS (Hyclone) and

1% penicillin-streptomycin. G0 arrest of MEF was achieved by starvation for 48 h in medium lacking

FBS. All MEF used were under passage 4, as after passage 4 Cyc E DKO MEF undergo a “replicative

crisis” (12941272).

3.6.2 Cell cycle synchronization

MOVAS were cell cycle synchronized by either 24 or 48 h serum starvation (serum-free DMEM

and 1% penicillin-streptomycin). 48 h serum starvation was used for most studies, although Western blot

analysis revealed that the additional 24 h of serum starvation did not significantly alter p27 expression

(data not shown). For cell cycle re-entry, MOVAS were given serum-restored (10% FBS) media.

3.6.3 In situ [Ca2+] manipulation

In order to manipulate intracellular calcium concentration, MOVAS were treated with the cell

membrane-permeable calcium chelator BAPTA-AM (Sigma, A1076) or the calcium ionophore

47

ionomycin (Sigma, I9657-1MG). For in situ analysis of calcium-sensitive p27 degradation in quiescent

MOVAS, G0-synchronized MOVAS were treated with 10 μmol/L BAPTA or 0.5 μmol/L ionomycin for

10, 20 or 30 min prior to protein extraction. For in situ analysis of calcium-sensitive p27 degradation in

proliferating MOVAS, G0-synchronized MOVAS were pre-treated with 50 μmol/L BAPTA or 0.5

μmol/L ionomycin for 30 min prior to serum stimulation and protein extraction.

3.6.4 In situ inhibition of CaMK-II, MEK and ubiquitin proteasome

For in situ analysis of CaMK-II-sensitive p27 degradation, G0-synchronized MOVAS were pre-

treated with 2 μmol/L of the cell membrane-permeable CaMKII inhibitor myristolyated-AIP (BIOMOL,

P-212) for 30 min prior to serum stimulation and protein extraction. For in situ analysis of MEK-sensitive

p27 degradation, G0-synchronized MOVAS were pre-treated with 10 μmol/L of the cell membrane-

permeable MEK 1/2 inhibitor U0126 (Cell Signaling, 9903) for 30 min prior to serum stimulation and

protein extraction. For in situ analysis of ubiquitin proteasome-sensitive p27 degradation, G0-

synchronized MOVAS were pre-treated with 10 μmol/L of the cell membrane-permeable ubiquitin

proteasome inhibitor MG-132 (Sigma, C2211) for 30 min prior to serum stimulation and protein

extraction.

3.6.5 Protein extraction

MOVAS cells were kept on ice, washed twice with PBS and resuspended with lysis buffer

containing 50 mmol/L C. Tris (pH 7.4), 250 mmol/L NaCl, 5 mmol/L EDTA, 0.1% NP-40, 100 mmol/L

DTT, 0.1 mmol/L Na3VO4, 2 mmol/L PMSF, 10% glycerol, 1% Phosphatase Inhibitor Cocktail 2

(Sigma), and Complete Protease Inhibitor (Roche). Cell suspensions were kept on ice and homogenized

with a sonicator (Fisher Scientific, Sonic Dismembrator Model 60) (2 pulses for 10 s at power 10, 2 min

between). Whole cell protein extracts were then isolated by collecting the supernatant after centrifugation

(1020 g, 10 min at 4 ºC). An aliquot was utilized for determination of protein concentration by BCA

quantification assay (Sigma, QPBCA-1KT). Whole cell protein extracts were stored at -80 ºC.

3.6.6 Calcium treatment of whole cell extracts

For initial analysis of calcium-sensitive p27 expression, MOVAS whole cell extracts were

incubated with Ca2+ (500 nmol/L), calmidazolium (500 ng per 25 μg protein), EGTA (5 mmol/L), water

or DMSO controls for 30, 60, 90, or 120 min at 37 ºC prior to p27 Western blot analysis.

3.6.7 Immunoprecipitation

For calmodulin immunodepletion, 500 μg MOVAS whole cell protein extract was tilt-mixed for

2 h at 4 ºC with 500 ng rabbit anti-calmodulin antibody (Santa Cruz, sc-5537) and lysis buffer to a total

volume of 1 ml. Immune complexes were collected by incubation with 60 μl GammaBind G Sepharose

Resin (GE Healthcare, Piscataway, NJ) for 2 h at 4 ºC. After centrifugation (10 000 rpm for 10 min at 4

48

ºC) the supernatant was collected as calmodulin-depleted protein extract. For immunoprecipitation of

calmodulin-binding proteins, 500 μg MOVAS whole cell protein extract was tilt-mixed with 100 μg

Calmodulin Sepharose 4B resin (GE Healthcare, 17-0529-01) and calcium binding buffer (lysis buffer

containing 2 mmol/L Ca2+) to a total volume of 1 ml for 4 h at 4 ºC. Unbound proteins were cleared by

three washes and centrifugations (10 000 rpm for 10 min at 4 ºC) with calcium binding buffer. Bound

proteins were eluted by incubation with 50 μl elution buffer (lysis buffer containing 2 mmol/L EGTA) for

5 min at RT, centrifugation and collection of supernatant.

3.6.8 Western blot

Either 15 (for 15-well) or 25 μg (for 12-well) of MOVAS whole cell protein extract was loaded

per lane of Novex 12% Tris-Glycine gel (Invitrogen, EC6008BOX or EC60085BOX). SDS-PAGE

electrophoresis was run using XCell Sure Lock Electrophoresis Cell chamber (Novex, San Diego CA)

and Power Ease 500 power supply (Novex) at 150 V, 50 mA and 25 W for 90 min. Samples were

transferred to a PVDF membrane (Perkin-Elmer, NEF1002001PK) at 30 V, 220 mA and 30 W for 90

min. Membranes were blocked for 1 h at RT with 5% non-fat dry milk in TBS-T. Membranes were

hybridized with primary antibodies (rabbit anti-p27 (Santa Cruz, sc-527), rabbit anti-p-p27(Thr187)

(Santa Cruz, sc-16324-R), rabbit anti-IQGAP1 (Santa Cruz, sc-10792) or rabbit anti-GAPDH (Santa

Cruz, sc-25778) in blocking buffer overnight at 4 ºC, washed 3 times with TBS-T, hybridized with goat

anti-rabbit IgG-HRP secondary antibody (Santa Cruz, sc-2054) in blocking buffer for 35 min at RT and

washed 3 times with TBS-T. Protein bands were detected with ECL reagents (Perkin-Elmer,

NEL104001EA) and developed with blue x-ray film (Perkin-Elmer, NEF596) and a medical film

processor (Konica Minolta, SRX-101A). Densitometry was performed using GS-800 Calibrated

Densitometer (Bio Rad, Hercules CA) and Quantity One v4.6.1 software (Bio Rad).

3.6.9 Coomassie staining

After SDS-PAGE electrophoresis, Tris-glycine gels were incubated with Coomassie Brilliant

Blue stain (Sigma, B-0770) overnight at RT. Gel was washed with Destaining Solution I (250 ml

methanol, 50 ml acetic acid, 200 ml H2O) and Destaining Solution II (25 ml methanol, 25 ml acetic acid,

440 ml H2O) until bands could be resolved.

3.6.10 Mass spectroscopy

Protein bands of interest on SDS-PAGE gel were identified by Coomassie staining. Bands were

cut from the gel using a razor blade and stored in 1% acetic acid solution for mass spectroscopy analysis

(Mass Spectroscopy Facility, Hospital for Sick Children, Toronto ON).

49


One-way ANOVA was followed by post-hoc Bonferroni’s test. Data shown are mean + SE.

Analyses were performed on Graphpad Prism v5.0 (GraphPad Software Inc, La Jolla, CA).

3.7 Results: Role of calcium/calmodulin on p27 degradation in vascular smooth muscle cells 3.7.1 Studies in whole cell protein extracts

Given preliminary data from our lab of increased Ca2+/CaM decreasing levels of p27, initial

experimental approaches aimed to determine the reproducibility of these findings. Replicating the

previous methodology, G0-synchronized whole cell extracts (WCE) of isolated proteins from an

immortalized mouse smooth muscle cell line (MOVAS) were incubated with physiological concentrations

of Ca2+/CaM and subsequent p27 Western blot analysis performed (Appendix 5). Several variations of

this original approach were performed, including the use of: 1) CaM immuno-depleted extracts

(Appendix 6), 2) a spectrum of incubation times to analyze calcium-sensitive effect on p27 degradation

over time (Appendix 7), 3) 4 h and 6 h serum-synchronized whole cell extracts to determine the effect of

cell cycle stage (Appendix 8 and Appendix 9), and 4) WT and cyclin E1-/-E2-/- (Cyc E DKO) mouse

embryonic fibroblasts (MEF) whole cell extracts to examine the role of cyclin E-dependence (Appendix

10). Unfortunately, collective findings from these studies were not able to reproduce original findings.

Results did not display significant differences in p27 levels due to Ca2+/CaM treatment.

The complexity of regulatory pathways surrounding p27 and relevant findings in the literature

suggest the potential for Ca2+/CaM-mediated p27 regulation in VSMC. However, it is also possible that

recently demonstrated findings are tissue-specific and that p27 degradation is regulated by calcium-

independent mechanisms in VSMC. Before drawing conclusions, it is important to note that studies thus

far have manipulated Ca2+/CaM in cell lysates, as per the original methodology. However, in whole cell

protein extracts, the integrity of the cell machinery has been altered and physiological processes may be

compromised. Therefore, it may not be the optimal system for analyses of p27 degradation.

3.7.2 Expression of p27 across the cell cycle in MOVAS

3.7.2.1 Characterization of cell cycle-dependent p27 degradation in MOVAS

Before proceeding with further investigations of the calcium-dependence of p27 degradation,

normal expression of p27 across the cell cycle was characterized by Western blot analysis in cell cycle-

synchronized MOVAS. Results show high levels of p27 during G0, with a near-complete absence of p27

by 8 h of serum stimulation (Fig. 13). Interestingly, at the 4 h serum time point, p27 levels are noticeably

decreased compared to G0, but remain plainly detectable. Therefore, 4 h of serum stimulation was

50

A

MOVAS

0 4 8 120.0

0.5

1.0

1.5

** *

Serum (h)

P27/

GAP

DH

P27

GAPDH

Serum (h)0 4 8 12

B

Figure 13. Expression of p27 across the cell cycle in MOVAS. MOVAS were cell cycle synchronized by 24 h serum starvation. Proteins were extracted every 4 h between G0 and 12 h serum and subsequent p27 Western blot analysis performed. 25 µg of protein were loaded per lane. (A) Representative blot is shown. (B) Average densitometry values show a significant decrease in p27 expression between quiescence and cell cycle entry in MOVAS (N=3, *P<0.05 vs G0 by one-way ANOVA and post-hoc Bonferroni’s test ).

Figure 13

51

identified as a time point of interest for analysis of p27 degradation in MOVAS, as p27 is actively being

degraded but is still detectable by Western blotting.

3.7.2.2 Characterization of cell cycle-dependent p27 Thr 187 phosphorylation in MOVAS

As p27 phosphorylation at Thr 187 is a necessary prerequisite to Skp2 ubiquitin ligase-mediated

degradation, phospho-p27 (Thr 187) is an important intermediate of p27 for analysis of degradation.

Therefore, Thr 187-phosphorylation of p27 was also examined across the cell cycle in MOVAS using a

specific phospho-p27 (Thr 187) antibody. Results show that phosphorylated p27 is detectable at G0, and

p27 phosphorylation increases by the 4 h time point (Fig. 14), demonstrating a inverse-relationship

between p27 phosphorylation and total p27 levels, and further highlighting the suitability of 4 h of serum

stimulation as a time point of interest for future studies.

3.7.3 In situ calcium analysis of p27 degradation in quiescent MOVAS

3.7.3.1 Increased intracellular calcium does not affect p27 degradation in G0-synchronized MOVAS in

situ

Compared to studies in cell lysates, it was determined that an in situ approach examining the

effect of increased intracellular calcium in cultured cells on p27 expression was both a more appropriate

method of study and critical to investigate. Therefore, G0-synchronized MOVAS were incubated with the

cell membrane-permeable calcium-chelator BAPTA-AM (BAPTA) or the calcium-ionophore ionomycin

prior to protein extraction and p27 Western blot analysis. Temporal analysis revealed that increased

intracellular calcium did not significantly accelerate p27 degradation over time compared to BAPTA-

treated or untreated controls in quiescent MOVAS (Fig. 15).

3.7.3.2 Increased intracellular calcium does not affect Thr-187 phosphorylation of p27 in G0-

synchronized MOVAS in situ

Moreover, consistent with total p27, increased intracellular calcium also did not affect Thr 187

phosphorylation of p27 over time (Fig. 16). Given that p27 is physiologically degraded during cell cycle

entry and not during G0, it is possible that integral p27 degradation machinery present in proliferating

cells is not present or active in quiescent cells. Increased intracellular calcium alone may not be enough to

initiate p27 degradation in quiescent cells, accounting for the lack of effect seen.

3.7.4 In situ temporal analysis of calcium-sensitive p27 degradation in proliferating MOVAS

Consequently, in situ analyses were repeated to include proliferating MOVAS, comparing cells

synchronized between G0 and 4 h serum stimulation- time points identified as appropriate for p27

degradation analysis by previous characterization of p27 expression across the cell cycle (Fig. 17).

Temporal analysis results showed that ionomycin-induced intracellular calcium increase causes an

average trend of enhanced p27 degradation, and significantly accelerated reciprocal p27 (Thr187)

52

A

B

Figure 14. Thr 187 phosphorylation of p27 across the cell cycle in MOVAS. MOVAS were cell cycle synchronized by 24 h serum starvation. Proteins were extracted every 4 h between G0 and 24 h serum and subsequent phospho-p27 (Thr 187) Western blot analysis performed. 25 µg of protein were loaded per lane.(A) Representative blot is shown. (B) Average densitometry values show a trend of increased p27 phosphorylation between quiescence and cell cycle entry in MOVAS (N=3, P=NS by one-way ANOVA).

Serum (h)0 4 8 12

p-P27 (Thr-187)

P27

MOVAS

0 4 8 120

50

100

150

200

Serum (h)

p-P2

7/P2

7

Figure 14

53

A

B

GAPDH

0 10 20 30 10 20 30 10 20 30 min

PBS BAPTA Ionomycin

P27

G0 MOVAS

0 10 20 30 400.0

0.5

1.0

1.5PBSBAPTAIonomycin

Time (min)

P27/

GAP

DH

Figure 15. In situ analysis of calcium-sensitive p27 degradation in quiescent MOVAS. MOVAS were G0-synchronized with 48 h serum starvation and treated with BAPTA (10 µmol/L) or ionomycin (0.5 µmol/L) for 10, 20 or 30 min prior to protein extraction. P27 Western blot analysis was performed. 25 µg of protein were loaded per lane. (A) Representative blot is shown. (B) Average densitometry values show ionomycin treatment did not cause significant differences in p27 degradation over time compared to BAPTA and untreated controls (N=3, P=NS by one-way ANOVA).

Figure 15

54

Figure 16. In situ analysis of calcium-sensitive p27 Thr 187 phosphorylation in quiescent MOVAS. G0-synchronized MOVAS were treated with BAPTA (10 µmol/L) or ionomycin (0.5 µmol/L) for 10, 20 or 30 min prior to protein extraction. Phospho-p27 (Thr 187) Western blot analysis was performed. (A) Representative blot is shown. (B) Average densitometry values show ionomycin treatment did not cause significant differences in p27 phosphorylation over time compared to BAPTA and untreated controls (N=3, P=NS, by one-way ANOVA).

G0 MOVAS

0 10 20 30 400

1

2

3

4

5PBSBAPTAIonomycin

Time (min)

p-P2

7/P2

7A

B

0 10 20 30 10 20 30 10 20 30 min

PBS BAPTA Ionomycin

P27

p-P27 (Thr-187)

Figure 16

55

P27

p-P27 (Thr 187)

GAPDH

G0 1 h 2 h 4 h

PBS

P27

p-P27 (Thr 187)

GAPDH

G0 1 h 2 h 4 h

BAPTA

P27

p-P27 (Thr 187)

GAPDH

G0 1 h 2 h 4 h

Ionomycin

A

Figure 17

56

B Figure 17

BAPTA

G0 1 h 2 h 4 h0

2

4

6

8

Cell Cycle Stage

P27/

GAP

DH

Untreated (PBS)

G0 1 h 2 h 4 h0.0

0.2

0.4

0.6

0.8

1.0

Cell Cycle Stage

P27/

GAP

DH

Ionomycin

G0 1 h 2 h 4 h0.0

0.5

1.0

1.5

2.0

Cell Cycle Stage

P27/

GAP

DH

BAPTA

G0 1 h 2 h 4 h0.0

0.5

1.0

1.5

2.0

Cell Cycle Stage

p-P2

7/P2

7

Untreated (PBS)

G0 1 h 2 h 4 h0.0

0.2

0.4

0.6

0.8

1.0

Cell Cycle Stage

p-P2

7/P2

7

Ionomycin

G0 1 h 2 h 4 h0.0

0.5

1.0

1.5**

Cell Cycle Stage

p-P2

7/P2

7

C

57

DP27 in situ MOVAS

0 1 2 3 4 50

1

2

3

4

5PBSBAPTAIonomycin

Serum Stimulation (h)

P27/

GAP

DH

P27 Phosphorylation in situ

0 1 2 3 4 50

2

4

6

8PBSBAPTAIonomycin


p-P2

7/P2

7

**

Figure 17. In situ temporal analysis of calcium-sensitive p27 degradation in proliferating MOVAS. MOVAS (passage 23) were cell cycle-synchronized by 48 h serum-starvation and pre-treated with BAPTA (50 µmol/L), Ionomycin (0.5 µmol/L) or an equal volume of PBS for 30 min prior to serum stimulation. Proteins were extracted at G0, 1, 2 and 4 h serum time points and p27 and p-p27 (Thr 187) Western blot analyses performed. (A) Western blots are shown. (B) Average p27 densitometry values. (C) Average p-p27 densitometry values. (E) Average p27 and p-p27 densitometry values from different treatment groups were compared over time by normalizing to G0 values (N=3, **P<0.01 by one-way ANOVA and post-hoc Bonferroni’s test).

Figure 17

58

phosphorylation over time, compared to untreated (PBS) and BAPTA controls. Therefore, in situ analysis

reveals that in contrast to quiescent cells, calcium-sensitive acceleration of Thr 187 phosphorylation of

p27 and p27 degradation can be shown in proliferating VSMC.

3.7.5 In situ cell cycle stage analysis of calcium-sensitive p27 degradation in proliferating MOVAS

In situ analysis of calcium-sensitive p27 degradation in proliferating MOVAS was also repeated

as a cell cycle stage analysis, separating analyzing each cell cycle time point and directly comparing

samples from different treatment groups vs. different cell cycle stages on one Western blot (Appendix

11). Results show that compared to controls, there is no significant p27 decrease due to ionomycin

treatment at G0 and 1 h of serum stimulation. However, by 2 h serum, ionomycin treatment causes a

marked decrease in p27 compared to untreated controls, an effect that remains at 4 h serum. Moreover, at

4 h serum, ionomycin-induced intracellular calcium increase causes a significant increase in Thr 187 p27

phosphorylation. Therefore, cell cycle stage analysis results validate temporal analysis findings of

enhanced p27 phopshorylation and degradation in response to ionomycin-induced intracellular calcium

increase. Collectively, in situ data support the hypothesis that increased intracellular calcium can lower

p27 levels through enhanced Thr 187 phosphorylation-dependent degradation in VSMC and demonstrate

the importance of intact cell physiology and cell cycle stage on this process.

3.7.6 In situ analysis of CaMKII/MEK/ubiquitin proteasome pathway of p27 degradation in

proliferating MOVAS

Having established the calcium-sensitivity of p27 degradation in MOVAS, subsequent

experimental approaches aimed to elucidate molecular pathways linking calcium signalling to p27

degradation. Provided recent findings showing calmodulin kinase-II (CaMK-II)/MEK/ubiquitin-

proteasome-dependent regulation of p27 degradation in adenocarcinoma cells93, we sought to determine if

this mechanism is cell type-specific or common to VSMC. Accordingly, in situ analyses were repeated

comparing p27 levels in untreated cells to cells treated with the cell membrane-permeable inhibitors of

target molecules.

3.7.6.1 In situ analysis of CaMKII-sensitive p27 degradation in proliferating MOVAS

In order to assess putative CaMKII-sensitivity of p27 degradation in VSMC, cell cycle-

synchronized MOVAS at 4 h serum were pre-treated with the cell membrane-permeable CaMKII

inhibitor myristolyated-AIP (Fig. 18). Compared to quiescent controls, myristolyated-AIP-treated cells

did not show a significant change in total p27 levels, whereas untreated cells showed an average trend of

p27 decrease between G0 and 4 h of serum. Moreover, at 4 h of serum, myristoylated-AIP treatment

causes a significant reduction in p27 (Thr187) phosphorylation compared to untreated controls. Taken

59

Figure 18

0

1

2

3

P27/

GAP

DH

PBS m-AIP

G0

PBS m-AIP

4 h

0.0

0.5

1.0

1.5

p-P2

7/P2

7***

PBS m-AIP

G0

PBS m-AIP

4 h

A

B

P27

p-P27 (Thr 187)

GAPDH

PBS m-AIP

G0

PBS m-AIP

4 h

Figure 18. In situ analysis of CaMKII-sensitive p27 degradation in proliferating MOVAS. MOVAS (passage 23) were cell cycle-synchronized by 48 h serum-starvation and pre-treated with the cell membrane-permeable CaMKII inhibitor myristolyated AIP (m-AIP) (2 µmol/L) for 30 min prior to serum stimulation. Proteins were extracted at G0 and 4 h serum time points and p27 and p-p27 (Thr 187) Western blot analyses performed. (A) Western blots are shown. (B) Average p27 and p-p27 densitometry values (N=3, ***P<0.001 by one-way ANOVA and post-hoc Bonferroni’s test).

60

together, these data indicate that calcium-sensitive p27 degradation in MOVAS is dependent on CaMK-II,

which may act as an important regulator downstream of calcium/calmodulin signalling.

3.7.6.2 In situ analysis of MEK-sensitive p27 degradation in proliferating MOVAS

To assess putative MEK/ERK-sensitivity of p27 degradation in VSMC, cell cycle-synchronized

MOVAS at 4 h serum were pre-treated with the cell membrane-permeable MEK inhibitor U0126 (Fig.

19). Interestingly, U0126-treatment did not significantly increase cell cycle-dependent p27 degradation

and phosphorylation, compared to untreated cells. Therefore, unlike CaMK-II, calcium-sensitive p27

degradation does not appear to be MEK-dependent in MOVAS, differing from the mechanism previously

characterized in human adenocarcinoma cells.

3.7.6.3 In situ analysis of ubiquitin proteasome-sensitive p27 degradation in proliferating MOVAS

Finally, to investigate if cell cycle-dependent p27 degradation is occurring through ubiquitin-

proteasome degradation in VSMC, 4 h serum synchronized MOVAS were treated with the ubiquitin-

proteasome inhibitor MG-132 (Fig. 20). Results show that MG-132 treatment appears to cause inhibition

of cell cycle-dependent p27 degradation, while untreated cells display an average trend of p27

degradation. Although p27 degradation appears to be inhibited by MG-132 treatment, cell cycle-

dependent increases in p27 (Thr187) phosphorylation remain intact, supporting (Thr187) phosphorylated

p27 as the intermediate form of p27 recognized and degraded via the ubiquitin proteasome in VSMC.

Lack of definitive results with MG-132 treatment may also indicate that p27 is degraded in a calcium-

sensitive manner by alternative proteases in VSMC, such as calcium-sensitive calpains.

Taken together, these results show that in vascular smooth muscle cells, the calcium-sensitive p27

degradation pathway potentially shares common elements with the pathway in human adenocarcinoma

cells. In proliferating MOVAS, p27 degradation appears to be similarly dependent on CaMK-II and the

ubiquitin-proteasome, but interestingly, not MEK/ERK signalling. Therefore, CaMK-II-induced

phosphorylation of MEK/ERK leading to (Thr187) p27 phosphorylation and degradation may be a tissue

or pathology-specific mechanism that is restricted to the colon or carcinoma cells. Moreover, this

mechanism was previously characterized using CaMK-II overexpression, as opposed to our method of

stimulating endogenous CaMK-II via increased intracellular calcium concentration. It is possible that the

mechanism shown in adenocarcinoma cells is dependent on CaMK-II overexpression, and may be not be

replicable in a physiological setting. Overall, these results provide a starting point for determination of

signalling pathways involved in p27 degradation downstream of intracellular calcium increase in VSMC.

61

Figure 19

0.0

0.2

0.4

0.6

0.8

1.0

p-P2

7/P2

7*

PBS U0126

G0

PBS U0126

4 h

0

1

2

3

4

P27/

GAP

DH

PBS U0126

G0

PBS U0126

4 h

P27

p-P27 (Thr 187)

GAPDH

PBS U0126

G0

PBS U0126

4 h

A

B

Figure 19. In situ analysis of MEK-sensitive p27 degradation in proliferating MOVAS. MOVAS (passage 23) were cell cycle-synchronized by 48 h serum-starvation and pre-treated with the MEK inhibitor U0126 (10 µmol/L) for 30 min prior to serum stimulation. Proteins were extracted at G0 and 4 h serum time points and p27 and p-p27 (Thr 187) Western blot analyses performed. (A) Western blots are shown. (B) Average p27 and p-p27 densitometry values (N=3, *P<0.05 by one-way ANOVA and post-hoc Bonferroni’s test).

62

Figure 20

0.0

0.1

0.2

0.3

0.4

p-P2

7/P2

7

DMSO MG-132

G0

DMSO MG-132

4 h

**

0

1

2

3

P27/

GAP

DH

DMSO MG-132

G0

DMSO MG-132

4 h

P27

p-P27 (Thr 187)

GAPDH

DMSO MG-132

G0

DMSO MG-132

4 h

A

B

Figure 20. In situ analysis of ubiquitin proteasome-sensitive p27 degradation in proliferating MOVAS. MOVAS (passage 21) were cell cycle-synchronized by 48 h serum-starvation and pre-treated with the ubiquitin proteasome inhibitor MG-132 (10 µmol/L) for 30 min prior to serum stimulation. Proteins were extracted at G0 and 4 h serum time points and p27 and p-p27 (Thr 187) Western blot analyses performed. (A) Western blots are shown. (B) Average p27 and p-p27 densitometry values (N=3, **P<0.01 by one-way ANOVA and post-hoc Bonferroni’s test).

63

3.8 Results: Identification of novel calcium/calmodulin-sensitive vascular smooth muscle cell cycle proteins 3.8.1 Broad survey of cell cycle differential calmodulin-binding proteins in MOVAS

In addition to cyclin E and p27, there may be other cell cycle proteins that are regulated in a

calcium-sensitive manner in VSMC. To identify novel calcium-sensitive cell cycle targets, a broad survey

of cell-cycle dependent CaM-binding proteins was conducted in MOVAS. G0 and 4h serum-synchronized

whole cell protein extracts were bound to a CaM-sepharose column, and the eluates underwent SDS-

PAGE electrophoresis and Coomassie staining. Bands differing noticeably between G0 and 4 h serum

were extracted for mass spectroscopy analysis.

3.8.2 Identification of IQGAP1 by mass spectroscopy

Gels revealed a band consistently expressed at G0 but not 4 h serum around 250KDa, which was

identified as IQGAP1 by mass spectroscopy (Fig. 21). This result suggests that IQGAP1/CaM binding

differs between G0 and 4h serum cell cycle time points in MOVAS.

3.8.3 IQGAP1 is uniformly expressed across the cell cycle in MOVAS

Moreover, Western blot analysis revealed that IQGAP1 is relatively uniformly expressed across

the cell cycle in MOVAS (Fig. 22). Therefore, differences in amounts of CaM-bound IQGAP1 are not

due to overall differences in protein expression between G0 and 4 h serum. IQGAP1 is a GTPase that is

known to directly bind to CaM and several different proteins in a Ca2+/CaM-dependent manner, such as

B-Raf, Cdc42 and F-actin but is not known to bind to any cell cycle proteins111-113. However, findings

show IQGAP proteins are involved in proliferation and cell cycle progression114, 115. Therefore, IQGAP1

may function as an intermediate regulator bridging Ca2+/CaM signalling to cell cycle control in VSMC.

Studies with IQGAP1 could elucidate novel calcium-dependent binding proteins or downstream targets

involved in cell cycle control in VSMC.

3.9 Discussion 3.9.1 Summary

In this study, we demonstrated that cell cycle-dependent degradation of p27kip1 can be enhanced

by ionomycin-induced intracellular calcium increase in vascular smooth muscle cells, but that increased

intracellular calcium alone is not sufficient to trigger p27 degradation in quiescent cells. Moreover, results

show that cell cycle-dependent p27 degradation appears to be affected by pharmacological inhibition of

CaMK-II and the ubiquitin proteasome, but not MEK/ERK signalling, potentially distinguishing the

calcium-sensitive p27 degradation pathway in vascular smooth muscle cells from the established

mechanism in human adenocarcinoma cells93. Moreover, findings of CaMK-II-dependent MEK/ERK

phosphorylation, p27 Thr187 phosphorylation and ubiquitin proteasomal degradation published by Li et.

64

4h

IQGAP1

G0 4h G0 4h G0

Figure 21. Differentially expressed CaM-binding proteins between G0 and 4 h of serum-stimulation in MOVAS. A representative gel is shown. Mass spectroscopy analysis of differentially expressed bands (outlined in red) revealed the band around 250kDa to be IQGAP1. The smaller MW band was non-specific. (N=2)

Figure 21

65

0 4 8 12 16 20 240.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75


IQG

AP1

/GA

PDH

IQGAP1

GAPDH

Serum (h)0 4 8 12 16 20 24

Figure 22. IQGAP1 expression across the cell cycle in MOVAS. MOVAS were cell cycle synchronized by 24 h serum starvation. Proteins were extracted every 4 h between G0 and 24 h serum and subsequent IQGAP1 Western blot analysis performed. Average densitometry revealed no significant differences in IQGAP1 expression at different cell cycle stages. (N=3, P=NS by one-way ANOVA).

Figure 22

66

al were dependent on CaMK-II overexpression in adenocarcinoma cells. Our findings may be more

physiologically-relevant, as we utilized an in situ approach of manipulating intracellular influx of

extracellular calcium to examine downstream effects on p27 degradation, as opposed to artificial

overexpression of target proteins.

3.9.2 Limitations

3.9.2.1 Pharmacological inhibitors

Pharmacological manipulation is an indirect approach of demonstrating involvement of target

molecules in signalling pathways. Inhibitors were assumed to produce their desired effects, but

performance was not validated by quantifying amount or activity of target molecules. Moreover, such

inhibitors may have non-specific actions on other intracellular pathways, potentially confounding results.

3.9.2.2 Cell cycle synchronization of MOVAS

Our initial characterization of p27 expression across the cell cycle revealed 4 h of serum as a time

point of interest in which p27 levels were significantly decreased compared to quiescence, but p27

expression was still detectable. However, in situ p27 analyses did not reproduce findings statistically-

significant p27 decrease between G0 and 4 h serum in untreated cells. This may be because of inconsistent

or inadequate cell cycle synchronization of MOVAS cells by our protocol between experiments. A longer

duration of serum-starvation may be necessary to ensure complete cell cycle synchronization in each

experiment. Moreover, the 4 h serum time point was our best estimate of when to capture decreased p27

between G0 and its disappearance at 8 h. However, the 4 h time point is just one frame of a moving

picture; a greater duration of serum-stimulation time points may be required to properly capture the

kinetics of cell cycle-dependent p27 degradation.

3.9.2.3 In vitro studies only

All experiments were performed in vitro using an immortalized vascular smooth muscle cell line

(MOVAS). Findings would be of increased physiological relevance if they were reproduced in primary

smooth muscle cells, or translated to in vivo models.


3.9.3.1 Further exploration of putative calcium-sensitive p27 degradation pathway

In situ analyses should be repeated utilizing improved methods of cell cycle synchronization and

a greater variety of cell cycle time points to validate previous findings. Statistically significant decrease in

p27 expression must be produced to compare to results observed by inhibition of target molecules. These

studies demonstrate that endogenous Thr187 phosphorylation and degradation of p27 appear dependent

67

on CaMK-II and the ubiquitin proteasome. It would be of interest to determine if these target molecules

are critically required for calcium-sensitive enhancement of p27 degradation by pre-treating VSMC with

myristoylated AIP or MG-132 prior to ionomycin and examining if CaMK-II/ubiquitin proteasome

inhibition is able to overcome ionomycin-induced acceleration of p27 degradation. Moreover, inhibitor-

based studies could be strengthened by reproducing results using loss-of-function/rescue approaches. For

instance, dependency of p27 degradation on CaMK-II could be examined in cells with CaM-knockdown

and compared to knockdown cells “rescued” with wild-type CaMK-II transfection. Finally, the

physiological significance of CaMK-II-dependent SMC proliferation could be assessed by administering

myristoylated-AIP post-carotid artery injury and determining if CaMK-II inhibition is able to

significantly in vivo SMC proliferation, reducing arterial thickening (this experiment has already been

performed with MG-132, displaying significant inhibition of intimal hyperplaisia116).

3.9.3.2 Increase quantitative resolution of analyses

Having established calcium-sensitivity of p27 degradation in MOVAS, subsequent experiments

could aim to increase the quantitative resolution of these findings. Given success with our in situ

approach, additional studies could be performed quantifying calcium influx by utilizing calcium

indicators such as FURA-2AM and fluorescent imaging. Quantified endogenous calcium influx and

calcium transients in response to ionophore/chelator treatment can then be correlated to levels of p27

protein expression. Furthermore, similar studies can be performed employing a range of

inhibitor/ionophore/extracellular calcium concentrations and treatment times to reveal dose-response and

temporal relationships. Enriching current findings with detailed quantitative data would provide deeper

insight into the mechanism and specificity of observed results.

3.9.3.3 Elucidation of remaining components of calcium-p27 pathway

Finally, after thoroughly confirming the effect of intracellular calcium concentration, CaMK-II

and the ubiquitin proteasome on cell cycle-associated p27 degradation, the remaining pathway

components should be pursued. Similar in situ studies could be performed focusing on candidate

molecules downstream of CaMK-II, upstream of ubiquitin protease or identified targets from the

literature, such as Skp2 ubiquitin ligase. Co-IP studies can then be used to tease out putative protein-

protein interactions between confirmed regulatory molecules involved in calcium-sensitive p27

degradation.

68

3.9.3.4 Putative cell cycle involvement of IQGAP1

Prior to further investigating putative cell cycle involvement of IQGAP1, IQGAP1/CaM binding

should be confirmed through co-IP studies. IQGAP1 Western blot analysis of whole cell protein extracts

from different cell cycle stages should then be examined. Methods used to identify IQGAP1 as a cell

cycle-differential CaM-binding protein could also be repeated with different cell types such as primary

VSMC and more cell cycle stages in order to identify other novel CaM-binding proteins involved in cell

cycle regulation.

3.9.4 Implications

3.9.4.1 Adding another layer of understanding to complex p27 regulation

The potential divergence of MEK/ERK-dependency on calcium-mediated p27 degradation

between adenocarcinoma and vascular smooth muscle cells constitutes an example of tissue-specific

differences in control of p27 expression. Moreover, the inability of intracellular calcium increase to affect

p27 degradation in quiescent cells demonstrates that cell cycle machinery present during G1-to-S phase

transition is required for calcium-sensitive p27 degradation. These findings add another of complexity to

p27 regulation, confirming the existence of cell type- and cell cycle stage-differential mechanisms of p27

degradation.

3.9.4.2 Targeting p27 degradation is an effective method of treating restenosis

3.9.4.2.1 Insufficiency of endogenous p27 activity in pathological smooth muscle cell proliferation

Uncovering calcium-sensitive mechanisms of p27 regulation in VSMC is clinically significant, as

p27 is a molecular target for potential treatment of proliferative vascular pathologies. Interestingly, it has

been shown that intimal thickening in p27-null mice does not significantly differ from wild-type mice

following arterial injury117. This may imply that p27 is not an important regulatory molecule in the

response to vascular injury, or that p27-null mice are able to compensate for lack of p27 with alternate

genes of redundant function. However, this could also indicate that endogenous p27 function is drastically

down-regulated in arterial SMC immediately following vascular injury, such that p27 levels in wild-type

mice are identical to p27-null mice. Moreover, SMC isolated from restenotic human vessels display

decreased p27 levels and a corresponding increase in cyclin E expression118. These findings demonstrate

the insufficiency of endogenous p27 activity to inhibit SMC proliferation in vascular disease. Therefore,

interventions that aim to boost or enhance endogenous p27 expression may be effective in preventing

pathological SMC proliferation.

69

3.9.4.2.2 Examples of effective p27 targeting for in vivo inhibition of smooth muscle cell

proliferation

Accordingly, experiments that restore p27 function in SMC post-arterial injury have

demonstrated therapeutic potential. Adenovirus-mediated p27 overexpession inhibits neointima formation

in rat109 and pig119 models of vascular injury. Both non-selective pharmacological inhibition of the

ubiqtuin proteasome116 and specific Skp2-silencing significantly abrogates SMC proliferation and

neointima formation in balloon rat carotid artery injury, which may be due to associated preservation of

p27 expression106. Therefore, manipulation of calcium-dependent, cell cycle-associated p27 degradation

may prove to be a therapeutic intervention of greater specificity and effectiveness for prevention of SMC

growth and division in proliferative vascular diseases.

70

CHAPTER 4. GENERAL DISCUSSION

4.1 Interpretation

4.1.1 Relationship of reported calcium-sensitive mechanisms in vascular smooth muscle cells

In this report, we have explored related calcium-sensitive mechanisms of cell cycle regulation in

VSMC on three different levels: we have (i) determined the clinical potential of an established

mechanism (CaM/cyclin E interaction), (ii) confirmed the existence of a putative mechanism (CaMK-

II/p27) and (iii) identified a target novel mechanism (CaM-IQGAP1). Importantly, demonstration of the

therapeutic promise of disrupting CaM-cyclin E interaction justifies further elucidation of the calcium-

sensitive p27 degradation pathway, IQGAP1 cell cycle involvement and other novel mechanisms.

4.1.2 Overall calcium handling in vascular smooth muscle cells

These novel findings valuably contribute to our knowledge of mechanisms through which

Ca2+/CaM signalling regulates cell cycle progression in VSMC, and possibly other cell types. Moreover,

elucidation of these precise calcium-sensitive mechanisms provides insight into how overall calcium

handling is distributed between cell cycle and contractile functions in VSMC.

4.2 Limitations

4.2.1 Potential calcium-sensitive cell cycle regulation in endothelial cells

A major limitation of current DES agents is endothelial toxicity and subsequent risk of

thrombosis. Therefore, treatments for restenosis and other proliferative vascular conditions that possess

specificity of action for SMC over EC would be of great clinical value. Unfortunately, the CBS peptide

did not overcome this deficiency, as experiments revealed similar anti-proliferative efficacy of TAT-

CBS-treatment between HA-SMC and EC in vitro. This may indicate ubiquitous presence of CaM-cyclin

E interaction in the endothelium as well as smooth muscle. Therefore, potential therapeutic agents based

on other established calcium-sensitive mechanisms of cell cycle regulation in VSMC could also

demonstrate anti-proliferative effects in EC.

4.2.2 Requirement of gene therapy approach for smooth muscle cell-specific delivery

Despite significantly decreased endothelial cell proliferation in vitro, TAT-CBS did not affect re-

endothelialization in vivo, which we speculate may be due to in vivo pluronic gel delivery to the

adventitial surface of the artery. However, as DES administration involves direct application of drugs to

the inner surface of the artery, TAT-CBS could harm the endothelial lining as a novel DES agent. Based

71

on data thus far, CBS and similarly developed peptides based on other calcium-sensitive cell cycle

mechanisms in VSMC may be effective treatments for proliferative vascular diseases if a gene therapy

approach is utilized to produce SMC-specific expression. However, gene therapy has several pitfalls, such

as inefficient gene transfer, adverse immune responses, and vector toxicity; as a therapeutic approach,

gene therapy may be far from standard clinical use120. Therefore, possible reliance on gene therapy as a

delivery method reduces the clinical potential of CBS and similar peptides.

4.2.3 Contribution of extracellular matrix, circulating progenitors to vascular disease pathologies

Unregulated medial VSMC proliferation is only one component of the injury response in

proliferative vascular diseases. Extracellular matrix accumulation is also a significant process that

contributes to pathological vascular remodelling121. Unfortunately, data shows that CBS peptide is not

effective in preventing collagen deposition post-carotid injury. Moreover, circulating bone marrow-

derived smooth muscle progenitor cells have also been shown to play a role in the development of

atherosclerosis, although findings are controversial122-124. Therapies based on calcium-sensitive

mechanisms of VSMC cycle control are directed at a single aspect of proliferative vascular diseases:

rapidly dividing VSMC of the blood vessel wall. Therefore, their clinical efficacy may be limited without

combined use of additional therapeutics that aimed at other factors such as extracellular matrix deposition

and circulating progenitor cells.

4.2.4 Cyclin E/cdk2-independent cell cycle progression

Both established calcium-sensitive cell cycle mechanisms thus far are based on the cyclin E/cdk2-

regulated G1-to-S phase progression in VSMC. Increased intracellular calcium activates CaM, which (i)

binds to cyclin E, activating the cyclin E/cdk2 complex, and (ii) causes CaMK-II-dependent ubiquitin

degradation of p27, which removes p27-induced inhibition of cyclin E/cdk2. These related mechanisms

emphasize the physiological importance of calcium-sensitive regulation of G1/S transition in VSMC, and

its suitability for therapeutic targeting. However, classic understanding of cyclin E/cdk2 requirement for

cell cycle progression has been challenged by recent studies demonstrating cyclin E/cdk2-independent

proliferation in cancer125. Therefore, CBS and similar therapies specifically targeting cyclin E/cdk2-

dependent G1/S progression may be ineffective if rapidly proliferating VSMC are able to divide in the

same manner.

72

4.3 Implications/clinical significance

4.3.1 New generation of drug-eluting stent agents based on calcium-sensitive cell cycle mechanisms

Results from investigations with the TAT-CBS peptide highlight its candidacy as a novel

therapeutic agent. These findings indicate the potential of similar approaches based on other established

calcium-sensitive mechanisms. For instance, an inhibitory peptide based on protein-protein interactions

that govern CaMK-II-dependent ubiquitin proteasomal degradation of p27 may prove to be an equally

effective agent. Characterization of this family of calcium-sensitive mechanisms of VSMC cycle control

could form the basis for a new generation of DES agents that are (i) anti-proliferative, (ii) less toxic, (iii)

possess increased transduction efficiency for VSMC over EC, and (iv) have increased selectivity for

rapidly proliferating VSMC.

4.3.2 CBS and similar agents as novel cancer therapy

Although similar anti-proliferative efficacy of TAT-CBS between SMC and EC, lowers its

therapeutic significance as a novel DES agent, proven anti-proliferative capacity in more than one cell

type does not necessarily diminish its overall clinical potential. Given the universality of calcium-

dependent cell cycle regulation across cell types and tissues, this finding may strengthen the physiological

significance of CaM-cyclin E interaction and other such mechanisms, by demonstrating existence outside

of SMC. Importantly, these results show putative CaM-cyclin E interaction can be effectively targeted by

TAT-CBS in other cell types. In fact, we have shown that TAT-CBS treatment decreases the proliferation

of several cancer cell lines (data not shown). Therefore, in addition to proliferative vascular diseases,

TAT-CBS and potential future peptides based on similar mechanisms may be effective novel therapeutic

agents for the treatment of cancer.

4.4 Future Directions

4.4.1 Narrowing down essential motifs

Pharmacological application of CBS and similarly developed agents would be improved by

decreasing the size of synthetic peptides. CBS is 22 amino acids long; shorter peptide sequences may be

more resistant to degradation, increasing peptide lifespan and potential duration of therapeutic action.

Preliminary studies employing TAT-conjugated, sequentially truncated versions of CBS reveal a putative

essential anti-proliferative region of 5 amino acids (16-20) (data not shown). Therefore, future

investigations with CBS and similarly developed peptides should be aimed at elucidating essential motifs

within peptide sequences that are capable of producing equal anti-proliferative effects in VSMC.

73

4.4.2 Translational testing of CBS and similar agents

Results demonstrating the therapeutic effects of TAT-CBS and potentially other similar peptides,

merit further investigation of their clinical potential. Given success with pluronic gel-mediated delivery in

mouse models of carotid artery injury, translational testing of TAT-CBS and related peptides should be

performed on: (i) SMC isolated from diseased human vessels, (ii) large animal models of proliferative

vascular disease, and (iii) animals treated with DES administrating TAT-CBS, in order to confirm

therapeutic benefit and optimize treatment design. If TAT-CBS and its counterparts are able to uphold

their demonstrated properties across several levels of translation, they may represent exciting novel

experimental therapeutics for clinical trials.

4.4.3 TAT-CBS in cancer

As preliminary data shows TAT-CBS can inhibit proliferation of several cancer cell lines, further

investigation of the clinical potential of TAT-CBS as a cancer treatment is warranted. Investigations

should be performed confirming the existence of CaM-cyclin E interaction and other established VSMC

cycle calcium-sensitive mechanisms in cancer cells in vitro. Given successful results, therapeutic testing

of CBS and similar peptides could progress to in vivo cancer models.

4.4.4 Investigation of other novel calcium-sensitive cell cycle mechanisms in vascular smooth muscle

cells

This body of work illustrates the physiological and clinical significance of examining calcium-

sensitive cell cycle progression in VSMC. Accordingly, in addition to cyclin E, p27 and IQGAP1, other

cell cycle targets should be probed for potential calcium-sensitivity. Approaches similar to the methods

used to identify IQGAP1 as a putative calcium-sensitive cell cycle regulator could be used to determine

other novel targets. Moreover, in addition to cyclin E, other cell cycle regulatory proteins have been

shown to bind directly to CaM (p21), or contain putative CaM binding sequences (cyclins A, B, D and H,

cdks 1, 2, 6 and 7, p16ink4a and p57kip2)29. These proteins should be similarly investigated for calcium-

sensitive activity, CaM-binding, and suitability for development of potentially anti-proliferative,

therapeutic peptides.

74

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APPENDIX

84

Appendix 1

0.0

0.2

0.4

0.6

0.8

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1.2R

elat

ive

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WT MEF Cyc E1/2 KO MEF

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A

NC

Serum CBS (1 mmol/L)NC (1 mmol/L)

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5

10

15

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25

30

35

40

% o

f S p

hase

** ** WTKO

+ + + + + ++ +

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i. CBS Treatment B

Serum CMZ (6 µmol/L)

05

101520253035404550

% o

f S p

hase

ii. CMZ Treatment

WTKO

+ + + + + +

**

**

85

Appendix 1

Appendix 1. CBS prevents serum-stimulated increase in cell number and S-phase entry in a cyclin E-dependent manner. (A) Number of WT- & Cyc E1/2 DKO-MEFs treated with peptides (1 mmol/L). Peptides were nucleofected into asynchronous MEFs, followed by MTT assay after 48 h (N=3). Measures were normalized to the WT no peptide group. *P<0.05 vs. no peptide. (B) Cell cycle analysis of WT- & KO-MEFs after peptide treatment (i) or calmidazolium (CMZ, non-selective CaM inhibitor) (ii). Treatments were performed on starved cells followed by either 24 h (peptides) or 20 h (CMZ) serum stimulation. Percentage of S phase population was calculated from 10,000 cells of each group (N=3, **P<0.01 by one-way ANOVA and post-hoc Student’s t-test). Data reproduced with permission from former PhD student Jaehyun Choi.

86

0

10

20

30

40IntimaMedia

% P

CN

A-p

ositi

venu

clei

F-127 only TAT-CBS-His TAT-NC-His

** **

Appendix 2. TAT-CBS-His decreases VSMC proliferation in vivo. Percentage of total PCNA-positive nuclei and PCNA-positive nuclei in the intima vs. media (N=4 mice in each group, **P<0.01 by one-way ANOVA and post-hoc Bonferroni’s test). Data reproduced with permission from former PhD student JaehyunChoi.

Appendix 2

87

Appendix 3

+

A

B

C

IgG NC CBS

Appendix 3. Carotid artery injury BrdU immunostaining. Mice received IP injections of BrdU (50 mg/kg) 17, 9 and 1 h before harvesting carotid arteries. Frozen sections of injured mouse carotid arteries from 3 different distances from the carotid artery bifurcation (A=200 μm, B=450 μm, C=700 μm) were immunostained using mouse anti-BrdU-Cy5-conjugated primary antibody or mouse IgG. Frozen sections of mouse small intestine were used as a positive control. Representative fluorescent microscopy images are shown. Significant BrdU staining was not observed in either the TAT-CBS or TAT-NC group.

88

Appendix 4

0.00

0.50

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2.50

3.00 IP: cyclin E1

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ativ

e H

1-K

inas

e Ac

tivity

** **

No peptide

CBS NC

Appendix 4. CBS inhibits calcium-sensitive CDK2 activity in VSMC. Histone H1 in vitro kinase assay on G1/S-synchronized mouse VSMC extracts at 0 or 500 nmol/L [Ca2+] in the presence of CBS or NC (100 µmol/L). For all in vitro kinase assay experiments, N=3 for each condition, with experiments repeated at least twice. Results were normalized to the kinase activity of the untreated group (data not known) and the untreated group at 0 nmol/L [Ca2+] (**P<0.01 vs. no peptide by one-way ANOVA and post-hoc Student’s t-test for multiple comparisons). Data reproduced with permission from former PhD student Jaehyun Choi.

0 500 0 500 0 500 nmol/L Ca2+

89

AP27

GAPDH

H2O EGTA Ca2+ CaM DMSOCMZ

BG0 MOVAS WCE

H2O EGTA Ca2+ CaM DMSO CMZ0.0

0.5

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GAP

DH

G0 MOVAS WCE

H2O EGTA Ca2+ CaM DMSO CMZ0.0

0.5

1.0

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2.0

p-P2

7/P2

7

C

Appendix 5. P27 Calcium/Calmodulin Analysis. Protein whole cell extracts were made from MOVAS that were G0-synchronized by 24 h serum starvation. Whole cell extracts were incubated with water (untreated control), EGTA (5 mmol/L), Ca2+ (500 nmol/L), CaM (500 ng), DMSO (control), or calmidazolium (CMZ) (100 µmol/L) for 1 h at 37 ºC prior to p27 Western blot analysis. 25 µg of protein were loaded per lane. (A) Representative blot is shown. (B) Average p27 densitometry values. (C) Average putative p27 phosphorylationdensitometry. The higher molecular weight p27 band is believed to be phosphorylated p27 (N=7, P=NS by one-way ANOVA).

Appendix 5

90

P27

GAPDH

EGTA Ca2+ Ca2+/CaM

EGTA Ca2+ Ca2+/CaM0.0

0.5

1.0

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2.0p-

P27/

P27

EGTA Ca2+ Ca2+/CaM0.0

0.5

1.0

1.5

2.0

2.5

P27/

GAP

DH

B

Appendix 6. P27 Calmodulin-Dependence Analysis. Protein whole cell extracts were made from MOVAS that were cell cycle-synchronized by 24 h serum starvation. Whole cell extracts were CaM-immunodepletedthen incubated with EGTA (5 mmol/L), Ca2+ (500 nmol/L) alone or Ca2+ (500 nmol/L)/CaM (200 ng) for 1 h at 37 ºC prior to p27 Western blot analysis. 25 µg of protein were loaded per lane. (A) Representative blot is shown. (B) Average p27 and putative p27 phosphorylation densitometry. The higher molecular weight p27 band is believed to be phosphorylated p27 (N=2, P=NS by Student’s t-test).

A

Appendix 6

91

G0 MOVAS WCE

0 50 100 1500.0

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1.5EGTACa2+/CaMCMZ

Time (min)

P27/

GAP

DH

*

G0 MOVAS WCE

0 50 100 1500.0

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2.5EGTACa2+/CaMCMZ

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"p-P

27"/

P27

AP27

GAPDH

Time0 30 60 120 30 60 120 30 60 120

EGTA Ca2+/CaM CMZ

B

C

Appendix 7

92

EGTA

0 30 60 1200.0

0.5

1.0

1.5

Time (min)

P27/

GAP

DH

CMZ

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0.5

1.0

1.5

Time (min)

P27/

GAP

DH

EGTA

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1.5

Time (min)

p-P2

7/P2

7

*

Ca2+/CaM

0 30 60 1200.0

0.5

1.0

1.5

2.0

Time (min)

p-P2

7/P2

7

CMZ

0 30 60 1200.0

0.5

1.0

1.5

2.0

2.5

Time (min)

p-P2

7/P2

7

D EAppendix 7

93

Appendix 7. P27 Temporal Calcium/Calmodulin Analysis. Protein whole cell extracts were made from MOVAS that were G0-synchronized by 24 h serum starvation. Whole cell extracts were incubated with EGTA (5 mmol/L), Ca2+ (500 nmol/L)/CaM (500 ng), or calmidazolium (CMZ) (100 µmol/L) for 30, 60 or 120 min at 37 ºC prior to p27 Western blot analysis. 25 µg of protein were loaded per lane. (A) Representative blot is shown. (B) Average p27 densitometry values over time. (C) Average putative p27 phosphorylationdensitometry over time. (D) Average putative p27 phosphorylation densitometry at each time point. (E) Average putative p27 phosphorylation at each time point. (N=3, *P<0.05 for panels B and E, P=NS for panels C and D by one-way ANOVA and post-hoc Bonferroni’s test).

Appendix 7

94

A

4 h MOVAS WCE

0 50 100 1500

1

2

3

4

5EGTA

Ca2+/CaM

Time (min)

P27/

GAP

DH

B

4 h MOVAS WCE

0 50 100 1500.0

0.5

1.0

1.5

2.0

2.5EGTA

Ca2+/CaM

Time (min)

p-P2

7/P2

7

P27

GAPDHTime0 30 60 90 120

EGTA Ca2+/CaM

30 60 90 120

C

Appendix 8

Appendix 8. P27 4 h Serum Calcium/Calmodulin Analysis. Protein whole cell extracts were made from MOVAS that were cell cycle-synchronized by 24 h serum starvation, then given serum for 4 h. Whole cell extracts were incubated with EGTA (5 mmol/L) or Ca2+ (500 nmol/L)/CaM (500 ng) for 30, 60, 90 or 120 min at 37 ºC prior to p27 Western blot analysis. 25 µg of protein were loaded per lane. (A) Representative blot is shown. (B) Average p27 densitometry values over time. (C) Average putative p27 phosphorylationdensitometry over time (N=6, P=NS by one-way ANOVA).

95

6 h MOVAS WCE

0 50 100 1500.0

0.5

1.0

1.5

2.0EGTA

Ca2+/CaM

Time (min)

P27/

GAP

DH

6 h MOVAS WCE

0 50 100 1500.0

0.5

1.0

1.5

2.0

2.5EGTA

Ca2+/CaM

Time (min)

p-P2

7/P2

7

P27

GAPDHTime0 30 60 90 120

EGTA Ca2+/CaM

30 60 90 120

A

B

C

Appendix 9. P27 6 h Serum Calcium/Calmodulin Analysis. Protein whole cell extracts were made from MOVAS that were cell cycle-synchronized by 24 h serum starvation, then given serum for 6 h. Whole cell extracts were incubated with EGTA (5 mmol/L) or Ca2+ (500 nmol/L)/CaM (500 ng) for 30, 60, 90 or 120 min at 37 ºC prior to p27 Western blot analysis. 25 µg of protein were loaded per lane. (A) Representative blot is shown. (B) Average p27 densitometry values over time. (C) Average putative p27 phosphorylationdensitometry over time. One-way ANOVA was applied (N=3, P=NS).

Appendix 9

96

A

Appendix 10

Time0 30 60 120 30 60 120 30 60 120

EGTA Ca2+/CaM CMZ

P27

GAPDH

Cyc E DKO MEF

Time0 30 60 120 30 60 120 30 60 120

EGTA Ca2+/CaM CMZ

P27

GAPDH

WT MEF

97

Appendix 10

BWT MEF WCE

0 50 100 1500.0

0.5

1.0

1.5

2.0EGTA

Ca2+/CaMCMZ

Time (min)

P27/

GAP

DH

WT MEF WCE

0 50 100 1500

2

4

6

8

Time (min)

p-P2

7/P2

7

Cyc E DKO MEF WCE

0 50 100 1500.0

0.5

1.0

1.5

2.0

Time (min)

P27/

GAP

DH

Cyc E DKO MEF WCE

0 50 100 1500

2

4

6

8

10

Time (min)

p-P2

7/P2

7

Appendix 10. P27 Cyclin E-Dependence Calcium/Calmodulin Analysis. Protein whole cell extracts were made from Cyc E DKO or WT MEF that were G0-synchronized by 24 h serum starvation. Whole cell extracts were incubated with EGTA (5 mmol/L), Ca2+ (500 nmol/L)/CaM (500 ng), or calmidazolium (CMZ) (100 µmol/L) for 30, 60 or 120 min at 37 ºC prior to p27 Western blot analysis. 25 µg of protein were loaded per lane. (A) Representative blot s are shown. (B) Average p27 and putative p27 phosphorylation densitometry over time (N=3, P=NS by one-way ANOVA).

98

AG0

P27

p-P27 (Thr 187)

GAPDH

PBS BAPTA Ionomycin PBS

1 h serum

p-P27 (Thr 187)

GAPDH

PBS BAPTA Ionomycin

2 h serum

4 h serum

P27

PBS BAPTA Ionomycin

p-P27 (Thr 187)

GAPDH

P27

PBS BAPTA Ionomycin

p-P27 (Thr 187)

GAPDH

P27

Appendix 11

99

BG0

0.00.10.20.30.40.50.60.70.80.9

P27/

GA

PDH

1 h serum

0.0

0.5

1.0

1.5

2.0

P27/

GAP

DH

2 h serum

0.00.51.01.52.02.53.03.54.04.55.05.5

P27/

GA

PDH

**

4 h serum

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

P27/

GA

PDH

******

PBS BAPTA Ionomycin PBS BAPTA Ionomycin


Appendix 11

100

C

**

****



4 h serum

0.00

0.25

0.50

0.75

1.00

p-P2

7/P2

7

2 h serum

0.00

0.25

0.50

0.75

p-P2

7/P2

7

1 h serum

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

p-P2

7/P2

7

G0

0.00

0.05

0.10

0.15

0.20

0.25

p-P2

7/P2

7

Appendix 11. In situ cell cycle stage analysis of calcium-sensitive p27 degradation in proliferating MOVAS. MOVAS were cell cycle-synchronized by 48 h serum starvation and pre-treated with BAPTA (50 µmol/L) or ionomycin (0.5 µmol/L) for 30 min prior to serum stimulation. Proteins were extracted at G0, 1, 2 and 4 h serum time points and p27 and p-p27 (Thr 187) Western blot analysis performed. (A) Western blots are shown. (B) Average p27 densitometry values. (C) Average p-p27 densitometry values.(N=3, *P<0.05, **P<0.01 , ***P<0.001 vs PBS by one-way ANOVA and post-hoc Bonferonni’s test).

Appendix 11

101

calcium-sensitive mechanisms in vascular smooth … · 2012. 11. 1. · 2.8.6 tat-cbs does not...

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