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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
(ii)
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.
(iii)
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.
(iv)
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
(v)
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
(vi)
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
(vii)
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
3.6.11 Statistical Analysis
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
(viii)
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 Future Directions
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
(ix)
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
(x)
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
(xi)
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
(xii)
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
2.7.17 Statistical Analysis
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
Media
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Figure 9
28
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29
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30
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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
Tissue Collagen Collagen I Collagen III
NC
CBS
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Un CBS
A
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otal
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lgen
I/III
Are
a
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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otal
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Figure 11
37
F-127 TAT-NCTAT-CBS
A
B
0102030405060708090
100
% E
ndot
helia
lizat
ion
F-127 TAT-CBS TAT-NC
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 Future Directions
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
3.6.11 Statistical Analysis
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
Serum Stimulation (h)
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
Serum Stimulation (h)
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 Future Directions
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
1.0
1.2R
elat
ive
OD
595
No pep. No pep.CBS CBS
WT MEF Cyc E1/2 KO MEF
NC
*
A
NC
Serum CBS (1 mmol/L)NC (1 mmol/L)
0
5
10
15
20
25
30
35
40
% o
f S p
hase
** ** WTKO
+ + + + + ++ +
+ +
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
1.00
1.50
2.00
2.50
3.00 IP: cyclin E1
Rel
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
1.0
1.5
2.0
P27/
GAP
DH
G0 MOVAS WCE
H2O EGTA Ca2+ CaM DMSO CMZ0.0
0.5
1.0
1.5
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
1.5
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
0.5
1.0
1.5EGTACa2+/CaMCMZ
Time (min)
P27/
GAP
DH
*
G0 MOVAS WCE
0 50 100 1500.0
0.5
1.0
1.5
2.0
2.5EGTACa2+/CaMCMZ
Time (min)
"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
0 30 60 1200.0
0.5
1.0
1.5
Time (min)
P27/
GAP
DH
EGTA
0 30 60 1200.0
0.5
1.0
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
PBS BAPTA Ionomycin PBS BAPTA Ionomycin
Appendix 11
100
C
**
****
PBS BAPTA Ionomycin PBS BAPTA Ionomycin
PBS BAPTA Ionomycin PBS BAPTA Ionomycin
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