1. health and fitness
2. disease

CHARACTERIZING THE EXPRESSION PROFILE OF
ANGIOGENIC PROTEINS AFTER ACUTE SPINAL CORD INJURY
by
Tsz Lui Michelle Ng
B.Sc., The University of British Columbia, 2008
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES
(Zoology)
THE UNIVERSITY OF BRITISH COLUMBIA
(Vancouver)
June 2012
© Tsz Lui Michelle Ng, 2012
Abstract
Spinal cord injuries (SCI) are one of the most physically and psychologically
devastating injuries one can survive. Despite decades of intense research effort, robust
therapeutic treatment for this catastrophic condition remains elusive. The nature of the
sequelae of SCI is characterized by progressive cell death in the injury penumbra, resulting in
further neurological impairments. The intricate relationship between the vascular and
nervous systems has become increasingly evident in many aspects of both normal
physiology, and various pathological conditions, including SCI. Vascular abnormalities play
a central role in the propagation of secondary damage after SCI.
The aim of this thesis is to further the understanding of the vascular changes that
occur after acute SCI. The endogenous expression of three angiogenic proteins:
Angiopoietin-1 (Ang1), Angiopoietin-2 (Ang2) and Angiogenin will be examined after acute
traumatic SCI. In the first study, the concentration of these proteins will be measured in a
temporal series of cerebrospinal fluid (CSF) samples after human SCI. In the second study,
the relative protein expression of Ang1 and Ang2 will be characterized in rat spinal cord after
SCI.
In human, Ang1 in CSF is not significantly different from non-SCI values after the
initial spike at 24 hours post-SCI. Ang2 in CSF shows a delayed but persistent increase
through the first 5 days post-SCI. In contrast, Ang1 in rat spinal cord decreases as early as 2
hours post-SCI, while low molecular weight Ang2 increases dramatically after SCI, from 2
hours to 3 days post-injury, peaking with a 13-fold elevation at 24 hours post-injury. These
findings represent the first description of these proteins in the acute SCI setting in human
CSF and rat spinal cord. The sustained elevation of Ang2 illustrates a possible mechanism by
ii
which reported vascular dysfunction and increases in blood-spinal cord-barrier (BSCB)
permeability occurs after SCI. The patterns of change reported between the two studies may
allude to the feasibility of using CSF as a biological proxy to future investigations into the
biochemical events which occur in the spinal cord after SCI, and guide the development of
pharmacologic treatments for this devastating condition.
iii
Preface
This thesis contains material that has been partly or wholly published in the following:
A version of chapter 1 has been published in [Ng, M.T.L.] and Kwon, B.K. (2011) Chapter 6:
Pharmacologic Treatments for Spinal Cord Injury. In Spine Trauma (2nd ed.): Zigler, J.E.,
Eismont, F.J., Garfin, S.R., and Vaccaro, A.R. Chicago, IL, American Academy of
Orthopaedic Surgeons, 2011. I am the primary author of this review chapter, which was
edited by BKK.
Versions of chapters 1 and 2 have been published in [Ng, M.T.L.], Stammers, A.T., and
Kwon, B.K. (2011) Vascular Disruption and the Role of Angiogenic Proteins after Spinal
Cord Injury. Translational Stroke Research, 2(4): 474 – 491. Samples were collected by the
clinical research team of the Combined Neurosurgical and Orthopaedic Spine Program
(CNOSP) at Vancouver General Hospital. I designed the research and participated in the
acquisition and processing of clinical samples. I conducted all the molecular experiments
with technical advice from ATS. I carried out the analysis and interpretation of the data, and
drafted the manuscript, which was edited by BKK. Ethics approval was provided by the
University of British Columbia Human Ethics committee under certificates H04-70584 and
H08-06673, and the clinical trial was registered on ClinicalTrials.gov (ClinicalTrials.gov
identifier: NCT00135278).
I designed and coordinated all of the research presented in chapter 3. I conducted all the
molecular experiments, analyzed the data, and drafted the manuscript. I performed all animal
iv
surgeries with technical assistance from JHTL and ST. I performed all post-surgical animal
care, euthanasia and sample collection with the technical advice from JHTL. CKL and FS
provided technical assistance and advice with the molecular assays. BKK edited the
manuscript. Ethics approval was provided by the University of British Columbia animal care
committee under certificate A10-0026.
v
Table of Contents
Abstract .................................................................................................................................... ii
Preface ..................................................................................................................................... iv
Table of Contents ................................................................................................................... vi
List of Tables .......................................................................................................................... ix
List of Figures ......................................................................................................................... xi
List of Abbreviations ........................................................................................................... xiii
Acknowledgements .............................................................................................................. xiv
Chapter 1: Introduction ........................................................................................................ 1
1.1
The History and Epidemiology of Spinal Cord Injuries ........................................... 1
1.2
The Spinal Cord ........................................................................................................ 2
1.2.1
Anatomy of the Spinal Cord ................................................................................. 2
1.2.2
Spinal Cord Vasculature and Perfusion ................................................................ 4
1.3
The Neurovascular Unit ............................................................................................ 8
1.3.1
The Coupling of Angiogenesis and Neurogenesis ................................................ 8
1.3.2
The Blood-CNS Barrier ...................................................................................... 10
1.4
Secondary Pathogenesis after Spinal Cord Injury .................................................. 14
1.4.1
Vascular Dysfunction after Spinal Cord Injury .................................................. 15
1.4.2
BSCB Breakdown after Spinal Cord Injury........................................................ 17
1.5
The Angiogenic Proteins......................................................................................... 19
1.5.1
Angiopoietin-1 .................................................................................................... 20
1.5.2
Angiopoietin-2 .................................................................................................... 26
1.5.3
Angiogenin .......................................................................................................... 29
vi
1.5.4
The Expression and Role of Angiogenic Cues outside the Vascular System ..... 30
1.5.5
Angiogenic Proteins as Treatment after Spinal Cord Injury ............................... 31
1.6
Research Objectives ................................................................................................ 32
Chapter 2: Changes in Angiogenic Proteins after Acute Human Spinal Cord Injury .. 35
2.1
Introduction ............................................................................................................. 35
2.2
Materials and Methods ............................................................................................ 36
2.2.1
Patient Enrollment and Clinical Evaluation ........................................................ 36
2.2.2
Sample Collection and Processing ...................................................................... 37
2.2.3
Molecular Analysis ............................................................................................. 37
2.2.4
Statistical Analysis .............................................................................................. 38
2.3
Results ..................................................................................................................... 38
2.4
Discussion ............................................................................................................... 48
2.5
Conclusions ............................................................................................................. 52
Chapter 3: Characterization of Ang1 and Ang2 Protein Expression after Acute Rat
Spinal Cord Injury................................................................................................................ 58
3.1
Introduction ............................................................................................................. 58
3.2
Materials and Methods ............................................................................................ 59
3.2.1
Animals and Housing Conditions ....................................................................... 59
3.2.2
Surgical Procedures ............................................................................................ 60
3.2.3
Tissue Collection ................................................................................................ 61
3.2.4
Western Blot ....................................................................................................... 61
3.2.5
Quantification and Statistical Analysis ............................................................... 63
3.3
Results ..................................................................................................................... 63
vii
3.4
Discussion ............................................................................................................... 78
3.5
Conclusions ............................................................................................................. 85
Chapter 4: Integrated Discussion and Research Conclusions ......................................... 87
4.1
Summary of Findings .............................................................................................. 87
4.2
The Role of Angiogenic Proteins in Vascular Disruption after Spinal Cord Injury 90
4.3
Implications for the Future...................................................................................... 93
4.4
The Translation Highway ....................................................................................... 95
4.5
Conclusions ............................................................................................................. 99
Bibliography ........................................................................................................................ 101
Appendices ........................................................................................................................... 125
Appendix A Supplementary Information from Chapter 2. ............................................... 125
A.1
Demographics and Medical Records of Human Subjects in Chapter 2. ........... 125
Appendix B Supplementary Methodology from Chapter 3. ............................................. 127
B.1
Determination of Protein Concentration ........................................................... 127
B.2
SDS-PAGE ....................................................................................................... 127
B.3
Antibodies Specificities .................................................................................... 130
viii
List of Tables
Table 2.1
Demographics of SCI patients enrolled in the current study. .............................. 39
Table 2.2
Demographics of non-SCI subjects enrolled in the current study. ...................... 40
Table 2.3
Expression of Ang1 expression in CSF after acute human SCI .......................... 47
Table 2.4
Expression of Ang2 expression in CSF after acute human SCI .......................... 47
Table 2.5
Expression of Angiogenin expression in CSF after acute human SCI ................ 47
Table 2.6
Summary of serum and CSF Ang1 values reported in the current study and in
literature. ................................................................................................................................. 54
Table 2.7
Summary of serum and CSF Ang2 values reported in the current study and in
literature. ................................................................................................................................. 55
Table 2.8
Summary of serum and CSF Angiogenin values reported in the current study and
in literature. ............................................................................................................................. 56
Table 3.1
Sample population of experimental groups presented in the current study. ........ 61
Table 3.2
Ang1 protein expression in rat spinal cord after acute SCI. ................................ 77
Table 3.3
65 kDa (high molecular weight) Ang2 protein expression in rat spinal cord after
acute SCI. ................................................................................................................................ 77
Table 3.4
Total Ang2 protein expression in rat spinal cord after acute SCI. ....................... 77
Table 3.5
25 kDa (low molecular weight) Ang2 protein expression in rat spinal cord after
acute SCI. ................................................................................................................................ 77
Table S.1
SCI subjects enrolled in human clinical trial. .................................................... 125
Table S.2
Non-SCI control subjects enrolled in human clinical trial. ............................... 126
Table S.3
Laemmli buffer preparation. .............................................................................. 127
Table S.4
Stacking and resolving gels for SDS-PAGE preparation. ................................. 127
ix
Table S.5
Running buffer preparation. .............................................................................. 128
Table S.6
Transfer buffer preparation. ............................................................................... 128
Table S.7
Tris-buffered saline with Tween-20 preparation. .............................................. 129
Table S.8
Blocking solution preparation. .......................................................................... 129
Table S.9
Antibody preparation. ........................................................................................ 130
x
List of Figures
Figure 1.1
Schematic representation of the vascular supply of the spinal cord. .................... 7
Figure 1.2
The interface of the neurovascular unit at CNS capillaries. ............................... 13
Figure 1.3
Protein sequence of Ang1 in rats. ....................................................................... 21
Figure 1.4
Schematic representation of Angiopoietin signalling in endothelial cells. ........ 25
Figure 1.5
Protein sequence of Ang2 in rats. ....................................................................... 27
Figure 2.1
Mean Ang1 protein levels in CSF and serum after acute human SCI. ............... 42
Figure 2.2
Mean Ang2 protein levels in CSF and serum after acute human SCI. ............... 44
Figure 2.3
Mean Angiogenin protein levels in CSF and serum after acute human SCI. ..... 46
Figure 2.4
A comparison of Ang1 and Ang2 protein expression in CSF after acute human
SCI. ......................................................................................................................................... 48
Figure 3.1
Representative image of Ang1 protein levels in rat spinal cord after acute SCI.64
Figure 3.2
Quantification of Ang1 protein expression in rat spinal cord after acute SCI. .. 65
Figure 3.3
Representative image of Ang2 protein levels in rat spinal cord after acute SCI.66
Figure 3.4
Quantification of 65 kDa (high molecular weight) Ang2 protein expression in rat
spinal cord after acute SCI. ..................................................................................................... 67
Figure 3.5
Quantification of total Ang2 protein expression in rat spinal cord after acute
SCI. ......................................................................................................................................... 68
Figure 3.6
Quantification of 25 kDa (low molecular weight) Ang2 protein expression in rat
spinal cord after acute SCI. ..................................................................................................... 69
Figure 3.7
Ang2 protein expression at 120 hours post-injury. ............................................ 70
Figure 3.8
Ang2 protein expression at 24 hours post-injury. .............................................. 70
Figure 3.9
Ang1 antibody tested on rat adult peripheral tissues and uninjured spinal cord. 71
xi
Figure 3.10
Ang2 antibody tested on rat adult peripheral tissues and uninjured spinal cord.
................................................................................................................................................. 72
Figure 3.11
Ang1 antibody tested by different SDS-PAGE protocols and on recombinant
human Ang1 and Ang2. .......................................................................................................... 73
Figure 3.12
Ang2 antibody tested by different SDS-PAGE protocols and on recombinant
human Ang1 and Ang2. .......................................................................................................... 75
Figure 3.13
Ang2 antibody tested on recombinant human Ang1 and Ang2. ...................... 75
Figure 3.14
The same membrane has be probed for Ang1, Ang2, and β-actin. .................. 76
Figure 4.1
Relative expression of Ang1 in human CSF and rat spinal cord after acute SCI.
................................................................................................................................................. 90
Figure 4.2
Relative expression of Ang2 in human CSF and rat spinal cord after acute SCI.
................................................................................................................................................. 90
xii
List of Abbreviations
Ang1
Angiopoietin-1
Ang2
Angiopoietin-2
AIS
ASIA impairment scale
ASIA
American spinal injury association
a.u.
Arbitrary units
BBB
Blood brain barrier
BSCB
Blood spinal cord barrier
CNS
Central nervous system
CSF
Cerebrospinal fluid
ELISA
Enzyme-linked immunosorbent assay
hpi
Hours post-injury
kDa
Kilo Dalton (1000 Da)
NPC
Neural progenitor cells
NVU
Neurovascular unit
PNS
Peripheral nervous system
rcf
Relative centrifugal force
SCI
Spinal cord injury/injuries
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SEM
Standard error of the mean
Tie
Tyrosine kinase with immunoglobulin (Ig) and epidermal growth
factor (EGF) homology domains
VEGF
Vascular endothelial growth factor
xiii
Acknowledgements
First and foremost, I would like to thank my graduate supervisor Dr. Brian Kwon for
giving me the opportunity to work with him during my graduate training. The best lessons in
life really are learnt through example. Thank you for the inspiration of a lifetime. Thank you
to all members of the Kwon lab, past and present. Jae Lee, I cannot overstate how important
and indispensible your presence has been throughout the years. Lisa Anderson, thank you for
the many ‘Lis-Adventures’. Craziness is purely a relative measure of genius as a function of
caffeine (or EtOH) intake.
Thank you to my committee members Drs. Wolfram Tetzlaff and Matt Ramer for
their valuable advice in not only science, but how to be a scientist. Thank you to all the
members of their laboratories for putting up with my constant harassment for advice and/or
equipment. The laughter I have shared here at ICORD far, far outweighs the seemingly
endless hours spent slowly grinding away at locked up writing in my office, running
molecular assays (thinking I would be a great chef after this life), or realizing I can now
communicate in the language of rats (or will now turn into rat-woman after my birthday rat
bite).
Thank you to my less nerdy friends who remained committed to our friendship
despite my physical absence throughout the years. I probably would not be here today
without those who willingly (or otherwise) lent an ear, a shoulder or gave a hug during the
tougher of times. Thank you to those who have volunteered (or were coerced into) reading
this thesis and/or were bombarded with the many versions of text that arose from it. To those
overseas, each and every experience we have shared together strengthens our friendship
beyond the ocean that separates us. EH, MH, YJ, KK, you girls are my lifesavers, literally!
xiv
Karen-san, I will always remember your favourite saying: “When your labs aren’t working,
you’re learning”.
Thanks to my family for being behind me during times of stress-induced crises and
their only-occasionally-wavering support in my seemingly never-ending journey through
school. And finally, to the many individuals with spinal cord injuries who live their lives
with such courage and strength, thank you for reminding me – every late night, weekend,
birthday and holiday that I am at the lab – why I am doing this.
xv
Chapter 1: Introduction
This introductory chapter will provide an overview of concepts that are important in
the discussion of this thesis work, and give the rationale and objectives of subsequent
chapters. The introduction will be presented in 6 sections. First, a brief history of spinal cord
injuries (SCI) and its epidemiology will be described. This will be followed by a basic
appreciation of the anatomy of the spine, spinal cord, and its vascular supply. Next,
interactions between the vascular and nervous systems will be considered in the context of
the neurovascular unit (NVU) and the blood-central nervous system (CNS) barrier. This will
be followed by a critical appraisal of the pathophysiology of SCI, highlighting the role of
vascular damage, and the breakdown of the blood-spinal cord-barrier (BSCB) in the
pathophysiology of SCI; as well as the endogenous angiogenic response after SCI. Finally, a
framework for the angiogenic proteins associated with this thesis work will be presented.
This chapter concludes with the objectives and rationales of the two studies which will be
addressed in this thesis.
1.1
The History and Epidemiology of Spinal Cord Injuries
Each year, over ten thousand North Americans suffer acute and permanent paralysis
after sustaining traumatic SCI [1]. SCI are not only one of the most physically disabling and
psychologically devastating traumas that an individual can survive, the socioeconomic
burden is enormous. Estimates of the annual medical and rehabilitative expenses are over $3
million for an individual with complete cervical cord paralysis [2, 3]. While historically this
has been an injury of the youth, an aging population prone to suffering SCI after falls has
altered the demographics of SCI, with a second peak appearing in the age distribution of
1
traumatic SCI in the elderly population aged 65 and above [4]. As our population continues
to age, the incidence and prevalence of SCI can be expected to rise.
SCI were first documented in 17th century, B.C., in the Edwin Smith papyrus, in
which it was deemed “a condition for which there is no ailment” [5, 6]. Centuries have
passed since the time when SCI were considered inescapably fatal, and advances in both
acute medical care and rehabilitation have most SCI patients with reasonably optimistic
prognoses. Life expectancy for an individual who suffered acute traumatic SCI at age 20, and
surviving the first 24 hours post-injury, is approximately 90% that of an age- and sexmatched individual without SCI [2]. Although SCI are now no longer consistently lifethreatening conditions, they are undoubtedly life-altering. The poor neurologic outcome for
SCI patients have prompted the development of a vastly expanding body of literature aimed
at understanding the massively complex pathophysiology of SCI. In the past four decades, a
plethora of therapeutic strategies have arisen, many of which have shown promise in the
laboratory setting [7-9], some of which have even successfully gone into human clinical trials
[10, 11]. However, despite these intense scientific efforts, primary functional outcomes from
these trials have largely been negative [12]; and to date, there is no single convincingly
efficacious treatment to improve neurologic recovery for this devastating condition.
1.2
1.2.1
The Spinal Cord
Anatomy of the Spinal Cord
Anatomically, the spinal cord is an extension of the brainstem that is housed within
the spinal canal. The spine is divided into 5 sections: cervical (C, from the bottom of the
brainstem to lower neck), thoracic (T, chest), lumbar (L, back), and sacral and coccygeal (S
2
and Co, lower back) regions. In human, the spinal cord runs down the spinal canal through to
approximately where the lumbar L1 and L2 vertebra are found, where it tapers off into the
conus medullaris [13]. The end of the spinal cord is continuous with the cauda equina, a
bundle of nerve roots that innervate the lower extremities. 31 pairs of spinal nerves connect
the CNS and the peripheral nervous system (PNS) at each of the 31 spinal levels (8 cervical,
12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal) [13]. Motor nerves extend out to innervate
and control skeletal muscles, while sensory nerves bring feedback from our skin, muscles,
joints, et cetera, to facilitate a proper response in accordance to the environmental stimulus.
The spinal cord in comprised of two main tissue types: white matter and grey matter.
In a spinal cord cross-section, these can easily be identified; with the grey matter appearing
in a distinctive ‘butterfly’ shape embedded within peripheral white matter. Grey matter
contains mostly neuronal cell bodies and axon terminals, and is where action potentials are
generated. White matter contains mostly myelinated axon tracts which relay these electrical
signals between the CNS and peripheral innervations targets.
The CNS is covered by three layers of meninges: dura mater, arachnoid mater, and
pia mater. The meninges enclose the CNS suspended in cerebrospinal fluid (CSF). CSF is a
clear liquid which baths the CNS, flowing at an average rate of up to 3 cm/s in humans [1416]. It is similar to blood plasma in terms of content, but with approximately 0.3% protein
concentration [17]. CSF insulates the CNS from systemic ionic changes, provides the
homeostatic environment required for efficient transmission of electrical signals between
neurons, and serves as a protective barrier, by providing neutral buoyancy for the brain and
spinal cord and preventing them from coming into contact with the skull and spinal canal,
respectively [18]. Moreover, because of its close proximity to the CNS, the CSF
3
compartment can provide indirect evidence of biochemical events occurring within the
parenchyma of the cord [19].
1.2.2
Spinal Cord Vasculature and Perfusion
The survival and function of cells within the spinal cord are dependent on the
transport of metabolites from vasculature. Perfusion of the spinal cord is largely dependent
on the arterial supply from the aorta [20] (Figure 1.1). Segmental radiculomedullary arteries
feed the anterior and posterior spinal arteries, which are the main circumferential arteries
outside the cord parenchyma, known as the ‘extrinsic’ arteries [21-25] (Figure 1.1). These
give rise to ‘intrinsic arteries’ which reside within the spinal cord parenchyma, and include
the central arteries as well as the pial plexus (Figure 1.1). The intrinsic arteries arborize into
extensive intramedullary arteriolar networks before ending as terminal capillary beds.
The intrinsic arteries within the spinal cord parenchyma can be separated into two
discrete circuits flowing in opposite directions [21-25] (Figure 1.1). This creates a watershed
region where terminal capillary beds of the two circuits overlap. The ‘centrifugal circuit’ is
fed by the anterior spinal artery, and supplies the central two thirds of spinal cord capillaries,
including the dense capillary networks which support spinal grey matter and the inner
regions of white matter [23]. The ‘centripetal circuit’ is fed by the posterior spinal arteries as
well as the many anastomoses arising from the anterior and posterior spinal arteries [23].
Vessels of this circuit supply much of the posterior white matter, dorsal horns, as well as
most of the peripheral white matter [21-24].
The centrifugal and centripetal vascular circuits meet in a complex network of
terminal capillary beds within the spinal cord. It is important to note that the density of such
4
capillary beds which serve the grey matter is approximately five times higher than those
which serve the white matter [23]. This is likely attributable to the greater metabolic
demands of cell bodies in the grey matter compared to axon tracts of the white matter [22,
26, 27].
5
6
Figure 1.1
Schematic representation of the vascular supply of the spinal cord.
(A) Longitudinal view of the major arterial supply of the spinal cord. (B) Cross-section view of the major
arterial supply of the spinal cord.
Segmental radiculomedullary arteries are fed by the aorta. The extrinsic arteries, the anterior and
posterior spinal arteries arise from the radiculomedullary artery. These extrinsic arteries arborize into a
complex network of intrinsic vessels which perfuse the spinal cord parenchyma.
A: aorta. R: radiculomedullary artery (segmental). ASA: anterior spinal artery. PSA: posterior spinal
artery. GM: spinal cord grey matter. WM: spinal cord white matter.
7
1.3
The Neurovascular Unit
In the CNS, vascular and nervous niches lie unavoidably together, not only in close
proximity, but in the way that they interact and rely on each other. CNS vasculature exists
within an elaborate matrix of nervous cells such as neurons and glia; but also vascular
components such as pericytes, astrocytes, vascular smooth muscle, as well as the
extracellular matrix. These components are integrated into what is now known as the NVU.
The NVU not only provides structural support, but also a means of communication and a way
to regulate homeostasis and metabolism for the proper functioning of the CNS.
The intricate relationship between the vascular and nervous systems has been
recognized for centuries. In recent years, vascular abnormalities have been described in
multiple neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS) [28-30],
Alzheimer’s disease [31, 32], and multiple sclerosis [33-36], often appearing even before
neurologic symptoms [33, 34, 37]. But whether these vascular changes are the cause or
consequence of the neurodegenerative condition remains unknown. Nonetheless, it is clear
that vascular components are not just passive bystanders supporting neurons, but active
players which adjust dynamically to meet the physical, metabolic, and functional demands
required by the nervous system.
1.3.1
The Coupling of Angiogenesis and Neurogenesis
As early as the 1543, renowned Belgian anatomist Andreas Vesalius depicted the
similar arborisation patterns of the vascular and nervous systems in De humani corporis
fabrica [38]. This apparent anatomical similarity spawns from the developmental plan of
these respective systems, and the actuality that a coordinated effort is required to dictate the
8
proper foundation for both systems. On a cellular level, migrating endothelial cell tips [39]
and axonal growth cones [40, 41] exhibit similar morphology. The cellular components of
the nervous network comprised of neurons with oligodendrocytes and Schwann cells
providing support for neurons, closely mimics that of endothelial cells in the vascular
network, and the supporting roles of pericytes and smooth muscle [39-41]. The relationship
between the vascular and nervous systems is tightly intercalated during development, in
quiescence, and after injury. Neovessels and neurites align with one another during
development [42-44] and share a variety of molecular cues guiding the migration and
maturation of nerves and vessels. In adult quiescence, large nerves depend on vascular
perfusion for nutrients and oxygen, while arteries require nervous signals to control
vasodilation or constriction
Vasculogenesis, the de novo formation of blood vessels from mesenchymal tissue is
distinct from the process of angiogenesis, which refers to the process of blood vessel growth
from existing vessels [45, 46]. Vasculogenesis precedes axon outgrowth, while angiogenic
outgrowth from the vascular plexus follows PNS sensory neurons [42, 43]. It has been
postulated that the primitive vascular plexus is formed first to provide metabolic support for
axons during their migration into the periphery, while the pruning and remodelling of the
vascular plexus into a mature vascular network is subsequently dependent on guidance cues
provided by sensory axons [43, 47].
The role of 4 major families of axon guidance cues in the developing vascular system
has been explored extensively (for review, see [48]), including the Slit/Robo family [49], the
Ephrins [50-53], the Semaphorins [54-59], and the Netrins [60]. Robo4 was reported to have
high specificity to vasculature [61], and to be chemotactic to endothelial cells [61, 62].
9
Sema3 [63, 64] and Netrins are chemotactic for endothelial tip cells [65-67]; while Ephrins
provide cues to set vascular boundaries in developing mice embryos [68-70]. Neuropilins, the
co-receptor for Semaphorin proteins, were found to also interact with vascular endothelial
growth factor (VEGF) [71, 72]. VEGF interacts with both neuropilin-1 [42, 71] and vascularderived Endothelin, which is expressed by smooth muscle cell, to guide sympathetic neurons
[44]. Interestingly, Semaphorin knock-out animals [73], like Netrin knock-out animals, both
show normal vascular phenotypes [74, 75], suggesting that the in vivo interactions between
vascular and nervous targets may be supplemented by multiple redundant targets, or
compensatory mechanisms.
1.3.2
The Blood-CNS Barrier
The intricate and elegant integration of the NVU is exemplified at the capillary level
by the cellular interface of the vascular and nervous systems at the blood-CNS barrier. In
1885, Ehrlich and Goldmann reported on a separation between peripheral circulation and the
CNS when each demonstrated that intravenous dye injections into peripheral circulation in
rabbits stained all tissues but the brain; while dye injected into the CNS did not stain
peripheral organs [76, 77]. Soviet physiologist Stern later proposed the concept of the bloodbrain-barrier (BBB) in her pioneering work describing the selective passage of substances
through the ‘barrière hémato-encéphalique’ [78]. However, it was not until 1967 that the
structural constituents of the BBB were located to the endothelial wall in CNS capillaries by
Reese and Karnovsky [79].
Anatomically, the blood-CNS interface is a selectively-permeable barrier manifested
by several components of the capillary wall including endothelial cells, pericytes, astrocytes,
10
and the extracellular matrix (Figure 1.2). Endothelial cells in the CNS are overlapping, with
tight junctions sealing paracellular spaces [80], no fenestrations [79-81], and minimal
pinocytosis [82]. They also have higher mitochondrial content than their counterparts outside
the CNS [83]. These mitochondria help to support the metabolically-expensive neurons and
glia of the CNS [83]. Molecularly, the junctional complexes between adjacent endothelial
cells are made of a combination of adherens and tight junctions. Adherens junctions are
found in all vessel walls. They mediate the adherence of endothelial cells to one another by
linking the actin cytoskeletons of adjacent cells [84]. Tight junctions are only found in bloodCNS interfaces. They are comprised of a complex of transmembrane proteins including
junctional adhesion molecules [85], occludins [86], and claudins [87], which span the entire
intercellular clef. Intracellular accessory proteins such as the Zonula Occludens (ZO) proteins
[88], link the transmembrane components to the cytoskeleton [89, 90].
Until recently, pericytes were a poorly characterized, heterogeneous group of cells
which have been observed in close proximity to endothelial cells [91-95]; their role assumed
to be a passive supporter for other vascular cells. Recent reports indicate that pericytes have a
significant role in the establishment and maintenance of the NVU [34, 92]. Pericytes were
first reported by Charles Rouget (and thus formerly dubbed the ‘Rouget cell’) in 1873 [96],
but it was not until 1923 that Zimmerman coined the term ‘pericytes’ for ‘peri’ – around, and
‘kytos’ – the Greek word for hollow vessels [97].
Pericytes serve several important functions as part of the NVU. Most apparent is its
role as part of the BBB. Pericytes are crucial to the establishment [98-100] and maintenance
of the BBB [101, 102]. They and act as a physical, metabolic, and transport barrier to
maintain CNS homeostasis and vascular quiescence. Other functions include vascular
11
development [103-108] and angiogenesis [106, 109-111] by influencing endothelial cell
proliferation, migration, and differentiation [98, 108], as well as mediating the formation of
vessels and the proper alignment of astrocytic foot processes against vessel walls [98]. They
express Glucose Transporter (Glut)-1 transporters [112] and serve an important role in
regulating CNS homeostasis by modulating blood flow by adjusting capillary diameter with
contractile elements [37, 113, 114].
Moreover, pericytes are known to migrate [110] in response to injury [115] or
hypoxia [106]. They exhibit multipotency, with an ability to differentiate to exhibit
characteristics of fibroblasts [115-117], endothelial cells [118], adipocytes [119],
chondrocytes [120], and immune cells [121, 122]. Changes in pericyte (and changes induced
by pericytes) have been reported to be implicated in multiple neurological pathologies
including both stroke [123, 124] and SCI [115].
The basal lamina, made of proteoglycan and laminin components, wraps around the
layer of endothelial cells and pericytes [34, 91, 92], providing physical support for the vessel
wall through interaction with other extracellular matrix components [125]. The basal lamina
can also stimulate the expression of tight junction-related proteins to help maintain BBB
function [126]. Astrocytic foot processes are juxtaposed against the basal lamina-covered
capillaries. These astrocytic processes serve as a critical route of communication between the
vascular and nervous systems. Like pericytes, they also have a critical role in the formation
and maintenance of the BBB [127-131]. Together, these components constitute a functional
NVU.
12
Figure 1.2
The interface of the neurovascular unit at CNS capillaries.
Tight junctions seal over-lapping endothelial cells. Pericytes surround the endothelial layer, and together,
these are ensheathed by the basement membrane. Astrocytic foot processes juxtapose CNS capillaries to
mediate communication between the vascular and nervous systems.
EC: endothelial cell. P: pericyte. TJ: tight junction. BM: basement membrane. A: astrocyte.
13
While the BSCB has slight structural and physiological differences from the BBB,
(some of which are discussed in [132]), functionally the two play similar roles in protecting
the CNS environment from systemic circulation [131]. There are a number of important
aspects to this function, which include: controlling ionic balance, regulating nutrient
transport, and restricting the passage of neurotoxic molecules and inflammatory cells into the
tenuous CNS.
The restrictive penetrance of the CNS is not limited to small metabolites and blood
proteins. The perception that the CNS is an immune privileged zone was first hypothesized in
1925 by Billingham and Boswell, who reported the lack of leukocyte infiltration in the brain
[133]. Absolute immune privilege of the CNS has since been disputed with increasing
evidence that the brain is indeed subjected to immunological surveillance (for review, see
[134, 135]). However, there is clearly a relative difference in immune privilege between the
CNS and peripheral tissues. The inflammatory response of the CNS under pathological
conditions propagates with a different mechanism and in a different timeframe than that in
peripheral tissues [136]. Furthermore, the number of immunological cells in the CNS is much
lower compared to the periphery (T-lymphocytes [137-139]; B-lymphocytes [140-142]; and
monocytes [136, 143]).
1.4
Secondary Pathogenesis after Spinal Cord Injury
It is now understood that when the spinal cord is injured, local mechanical forces
disrupt the complex vascular and cellular architecture of the cord, but rarely transect the cord
completely. This mechanical ‘primary injury’ is rapidly followed by an expanding cascade of
‘secondary damage’ mediated by pathophysiological mechanisms including ischemia,
14
excitotoxicity, inflammation, and oxidative stress. Attenuating these mechanisms to
minimize secondary damage and afford ‘neuroprotection’ to regions of the spinal cord that
have escaped the primary injury remains a principal therapeutic strategy.
1.4.1
Vascular Dysfunction after Spinal Cord Injury
It has been recognized for many years that trauma to the spinal cord causes immediate
vascular disruptions at the injury epicentre [144-148]. In 1911, Allen described the
development of hemorrhage and edema within the spinal cord after experimental SCI in dogs
and postulated the secondary injury theory, stating that progressive damage to the spinal cord
continues after the initial impact [145, 149].
Most of the necrotic damage to endothelial cells occur during the first 24 hours postinjury, and can largely be attributed to the initial mechanical insult [148]. This damage
primarily affects the microvasculature, with vascular abnormalities observed at the injure
epicentre as early as 5 minutes post-injury [150]. Ruptured vessels at the injury epicentre
results in petechial hemorrhage [148, 150, 151], which starts near the central canal and is
initially confined to the capillaries of the grey matter. The hemorrhage spreads to the white
matter by 2 hours post-injury [145, 152, 153]. Further endothelial cell loss after the first day,
manifested as a decrease in intact blood vessel staining by Rat Endothelial Cell Antigen
(RECA)-1 [154, 155] and Platelet Endothelial Cell Adhesion Molecule (PECAM) [156], is
mainly attributed to apoptosis triggered by ischemia [148]. Vessel density continues to
decrease during the first 2 days, with little or no observable vessels at the injury epicentre
[154, 156]. Vascular abnormalities remain even up to 9 months post-injury in human SCI
[157].
15
After SCI, angiogenic sprouting from vessels that were spared from the primary
insult, starts 3 [151] to 4 days post-injury [154], and is observed up to 1 week post-injury
[154]. Revascularization to an extent that is comparable to control values [156], or even up
several folds (540% increase in vessel density) [151], has been reported at 7 days post-injury
[151, 156]. However, these neovessels, which grow longitudinally through the injury
epicentre [154], are not associated with neurons, astrocytes [154], or pericytes [115]. Given
the important role that astrocytes and pericytes have on vascular function within the CNS,
this may indicate that although there is significant angiogenic outgrowth during the early
post-injury phases of SCI, these neovessels may not be fully functional.
The restoration of Glut-1 molecules, which are responsible for transporting a constant
supply of glucose across the BSCB to metabolically fragile CNS neurons, has not been
observed until the 2 weeks post-injury [156]. This further suggests that new neovessels may
not be fully capable of delivering nutrients (or therapies) to the injury site. Perhaps as a
consequence of the lack of integration of these neovessels into a functional NVU, there is
subsequent pruning of these vessels at 2 weeks post-injury [154]. There is a more prolonged
phase of angiogenesis from 4 weeks to 2 months post-injury, along with significant
deposition of new basal lamina [155]. This suggests that maturation and organization of
neovessels does not occur during the first angiogenic phase, and that much of the endothelial
cell sprouts are pruned away, with only a portion remaining to become stable, functionally
integrated blood vessels.
The disruption of local microvasculature also has profound effects on local blood
flow and perfusion. There is immediate vasospasm at the injury epicentre [158-162],
resulting in reduced perfusion to the remaining cord parenchyma [163-168]. Neurons have
16
high metabolic requirements [27, 169, 170], and are thus extremely vulnerable to reductions
in perfusion and resultant periods of ischemia. At rest, neurons require more than three times
the energy usage (in terms of ATP) than glial cells [170]. This difference increases to more
than five times per second in a neuron that is generating action potentials [170]. This may be
exacerbated by the loss of auto-regulatory mechanisms [163, 171-174] and systemic
hypotension [175-179], which are common in acute SCI patients, as the result of
hypovolemic and/or neurogenic shock [163, 171, 172, 180, 181]. Together, these result in the
loss of spinal cord microcirculation [158, 182, 183], impairing axonal conductance [184].
The progressive death of neurons in the hours, days, and even weeks after SCI has been well
documented [145, 149, 153, 162, 185-187].
1.4.2
BSCB Breakdown after Spinal Cord Injury
The conceptual integration of the vascular and nervous system is essential in
understanding the pathophysiology of SCI and provides a basis for potential intervention
strategies targeting both vascular and nervous systems. It is important to realize that the
consequences of the aforementioned microvascular damage are not limited to endothelial cell
death and the loss of perfusion, but also involves the breakdown of the BSCB. There is
extensive breakdown of the BSCB after SCI [152, 164, 188-192]. The lack of such a barrier
protecting the spinal cord allows for the indiscriminate passage of cellular toxic molecules
such as calcium [146, 193], excitatory amino acids [194, 195], free radicals [196],
erythrocytes [144-146, 152, 157, 188, 197], and inflammatory mediators [198] into the injury
penumbra, all of which may exacerbate secondary injury after SCI. Although BSCB
dysfunction has been reported largely from 2 [152, 164, 188, 190] to 4 weeks post-injury
17
[191], chronic abnormalities have been observed at 8 weeks in a mouse model of SCI [199],
and even 7 months post-injury in cats [189].
The time course of BBB and BSCB breakdown in multiple sclerosis [200-203],
Alzheimer’s disease [31, 204, 205], after traumatic brain injury (TBI) [206, 207], stroke
[205, 208] or SCI [198], have all been reported to closely parallel the progression of neuroinflammation. Accumulation of immune cells has been reported in perivascular spaces where
basement membrane and astrocytic foot processes were displaced after SCI [199], suggesting
that BSCB breakdown may be involved in the expansion of the post-injury inflammatory
response after SCI.
In animal studies, BSCB breakdown after SCI is manifested as the extravasation of
systemically-administered vascular tracers into spinal cord parenchyma. This has been
reported as early as 1 hour post-injury and remains elevated for at least 24 hours [144, 152,
156, 192]. This early peak in vascular leakage coincides closely with the acute inflammatory
response [198], implicating the role of vascular permeability in the propagation of the
inflammatory response after SCI. Increased BSCB permeability has also been observed
between 3 and 7 days post-injury in various models of SCI [156, 164, 188, 190, 199],
correlating with the initiation of angiogenesis and revascularization of the injury epicentre
[147, 148, 151, 154-156]. The destabilization of existing vessels increases vascular plasticity
and is necessary for angiogenic remodelling to occur. This is evident as a breach of tight
junctions, displacement of astrocytic foot processes, and separation of the basement
membrane [152, 188, 190, 209]. In the chronic phase of injury, up to 5.5 months, overlapping
endothelial cell junctions are reformed [188], although the perivascular space continues to
expand, with misaligned extracellular matrix, collagen layers, and displaced astrocytic foot
18
processes [188]. This suggests that despite endogenous reparative efforts, there are chronic
morphological abnormalities in the BSCB after SCI.
Opening of the BBB allows for the propagation of inflammation in and around the
injury epicentre. In response to the injury and to contain this area of inflammation, the glial
scar, which is inhibitory to neuronal growth, is created [210-212]. Interestingly, although the
glial scar has historically been labelled as arising from reactive astrogliosis [213], a recent
study has shown a significant role of pericytes in the formation of this scar, in addition to its
effects on BSCB physiology [115].
1.5
The Angiogenic Proteins
Given the implications of vascular disruption on secondary injury, the mechanisms by
which angiogenesis occurs and the BSCB restored are particularly relevant to the topic of
how neuroprotection can be achieved in acute SCI. In the next section, I will discuss the role
of three angiogenic proteins – specifically Angiopoietin-1 (Ang1), Angiopoietin-2 (Ang2),
and Angiogenin – in the regulation of angiogenesis and the restoration of the BSCB after
SCI.
As detailed in section 1.3.1, vascular growth factors have established roles in the
nervous systems that parallels and complements their effects in the vascular system. VEGF
was the first characterized vascular endothelial growth factor [214], characterized in 1983 for
promoting angiogenesis and endothelial permeability [215, 216]. It was also the first to have
reported neurotrophic effects [217-219]. Like VEGF, the Angiopoietins are a family of
growth factors that promote angiogenesis and regulate vascular permeability. Ang1 and Ang2
are the best-characterized members of the Angiopoietin family, and are essential for the
19
induction, maturation, and maintenance of blood vessels. Angiogenin is a potent endothelial
mitogen that belongs to the ribonuclease (RNase) A superfamily of ribonucleolytic proteins.
It is been heavily implicated in the pathogenesis of ALS by affecting neuron survival [220222].
1.5.1
Angiopoietin-1
Ang1 was isolated by Davis et al in 1996 [223] as a ligand for Tyrosine kinase with
Immunoglobulin (Ig) and Epidermal Growth Factor (EGF) homology domains (Tie) 2
receptors, which are predominantly expressed on endothelial cells [224, 225]. Ang1 is a 498
amino acid glycoprotein of approximately 70 kDa [223, 226] (Figure 1.3). The Ang1 protein
consists of 3 distinct domains: a short amino terminus which forms a ring-like structure to
super-cluster Ang1 homodimers together [227], a coiled-coil domain which mediates the
formation of Ang1 homodimers via a disulphide bond at Cys245 [227, 228], and the carboxyl
terminus which is homologous to the carboxyl terminus of fibrinogen, hence it being named
the fibrinogen-like domain [227, 228]. This domain is responsible for ligand activity [228]
and contains the binding site to Tie2 [227, 229].
20
Figure 1.3
Protein sequence of Ang1 in rats.
Red underlined sequence shows the antibody (which is used in chapter 3) recognition site. Yellow
highlights denote glycosylation sites (total of 5 sites). Green highlights denote MMP-2 cleavage
recognition sites (total of 19 sites). Turquoise highlights denote MMP-9 cleavage recognition sites (total of
3 sites).
21
Ang1 is constitutively expressed at a low basal level in quiescent adult vasculature
[223, 230-233] by perivascular mural cells such as pericytes [234-236] and smooth muscle
[233, 237]. Upon activation of its receptor Tie2, Ang1 induces pro-survival and antiapoptotic effects on endothelial cells [238-241] (Figure 1.4). Ang1 binding induces autophosphorylation of Tie2, which activates downstream Phosphotidylinositol 3-kinases (PI3K)
and Protein Kinase B (Akt) [233, 239]. This has numerous downstream pro-survival effects,
including the up-regulation of Survivin [239, 242-244], mammalian Target of Rapamycin
(mTOR) [245], and the inhibition of caspases 3, 7, 9, B-cell Lymphoma (Bcl)-2-associated
Death Promoter (BAD), Second Mitochondrial-derived Activator of Caspases (Smac) [239,
244, 246], and Forkhead Box class O (FOXO)-1 [243]. In addition, Ang1 has been shown to
interact with integrin to promote survival through a similar activation of Akt as well as
various mitogenic protein kinases [242, 247]. Ang1 also modulates other survival signals
such as Extracellular Signal-Regulated Kinases (ERK1/2) [245, 248], Stress Activated
Protein Kinase (SAPK), and c-Jun N-terminal Kinases (JNK) [248].
In addition to the role that it plays in promoting endothelial cell survival, Ang1 is
crucial in maintaining vessel quiescence by limiting vascular permeability and controlling
BSCB integrity [231] (Figure 1.4). Ang1 strengthens paracellular interactions and reduces
the number and size of endothelial gaps [249] by inducing the expression of adhesive
PECAM-1 [250] and tight junction proteins occludin [234] and ZO-2 [251]. Ang1 further
reinforces vessel integrity by inhibiting the transcription of genes associated with vessel
destabilization and remodelling [243]. By securing paracellular junctions, Ang1 effectively
limits the progression the inflammatory response by restricting the passage of inflammatory
cells from the bloodstream to CNS tissue [252]. Moreover, Ang1 activation of PI3K and Akt
22
[253, 254] inhibits the expression of inflammatory cytokine Nuclear Factor Kappa-lightchain-enhancer of activated B cells (NFκB) [255] and adhesion molecules Intercellular
Adhesion Molecule (ICAM)-1, Vascular Cell Adhesion Molecule (VCAM)-1, and E-selectin
[250, 254], which are required for the migration of inflammatory cells (Figure 1.4).
Although Ang1 has little to no proliferative effects on endothelial cells [232, 256,
257], it is essential in the later stages of angiogenesis, including the migration and
organization of vessel components into mature, stable vessels [231]. Ang1 mediates the
migration of vascular components to sites of angiogenesis, and the organization of these
components into tubule-like structures. This is mediated by the activation of Tie2 [258],
PI3K [259] and mTOR [245] resulting in the release of Matrix Metalloproteinase (MMP)
[259], the inhibition of Tissue Inhibitor of Metalloproteinases (TIMP), Focal Adhesion
Kinase (FAK) [259], and Nitric Oxide Synthase (NOS) [260, 261], all of which are known to
modulate cytoskeletal dynamics.
It has been hypothesized that the ability of Ang1 signalling to both maintain the
vasculature in a quiescent state as well as mediate angiogenesis may be attributed to
differential ligand-receptor complexes that Ang1 and Tie2 receptors form in mobile versus
confluent cells [262-264]. When associated with confluent, mature endothelial cells, Ang1
mediates the formation of trans-associated Tie2 homotypic paracellular complexes to
reinforce vascular integrity [262-264] (Figure 1.4). In contrast, when associated with mobile
endothelial cells, Ang1 associates and binds to the extracellular matrix [265] via β1-integrin
[266], and releases adhesion molecules which promote cell motility and migration of vessel
components [262, 263] (Figure 1.4).
23
24
Figure 1.4
Schematic representation of Angiopoietin signalling in endothelial cells.
(A) Ang1 binding to Tie2 promotes vascular integrity by securing cell-cell interactions and via
transcription of tight junction proteins.
(B) Ang1 binding to Tie2 receptors induces the activation of PI3K and Akt, via various mechanisms to
promote survival effects on endothelial cells.
(C) Ang1 binding to Tie2 receptors suppresses expression of NFκB and adhesion molecules to elicit antiinflammatory effects on endothelial cells.
(D) Ang1 also interacts with laminin to induce migration of endothelial cells.
(E) Ang2 exerts antagonistic effects by binding to, but not activating the Tie2 receptor to induce
downstream effectors.
EC: endothelial cell. P: pericyte. N: nucleus. WP: Weibel-Palade body. TJ: tight junction. ECM:
extracellular matrix.
25
1.5.2
Angiopoietin-2
Ang2 was first characterized in 1997 by Maisonpierre et al as the natural antagonist
of Ang1 [230]. While Ang1 is expressed at low basal levels constitutively, Ang2 expression
is more actively regulated to modify and counteract Ang1 signalling. Ang1 and Ang2 share
approximately 60% homology in their amino acid sequence (Figure 1.5), as well as a
common protein structure consisting of an amino terminal that modulates super-clustering, a
coiled-coil domain for the formation of homodimers, and a fibrinogen-like domain with
receptor-binding and ligand activities [227, 230, 267]. Ang1 and Ang2 bind to the same
domain on Tie2 with similar affinities [230, 267, 268] and conformation [229]. However,
they have opposite effects on receptor phosphorylation and activation (Figure 1.4).
26
Figure 1.5
Protein sequence of Ang2 in rats.
Red underlined sequence shows the antibody (which is used in chapter 3) recognition site. Yellow
highlights denote glycosylation sites (total of 6 sites). Green highlights denote MMP-2 cleavage
recognition sites (total of 34 sites). Turquoise highlights denote MMP-9 cleavage recognition sites (total of
3 sites).
27
While binding of Ang1 to Tie2 induces receptor auto-phosphorylation and triggers
downstream intracellular signalling pathways, the binding of Ang2 does not [228] (Figure
1.4). The differential effects that Ang1 and Ang2 have on receptor activation have been
hypothesized to be due to their different abilities to form homotypic oligomers. Native Ang1
is largely found in superclusters of tetramers or higher order oligomers, whereas native Ang2
is predominantly reported as homodimers [228, 269]. Receptor tyrosine kinases such as Tie2
often require multimerization for receptor activation [228, 269]. Thus it is conceivable that
although Ang2 dimers are able to bind to Tie2 receptors, they may not be sufficient to elicit
receptor auto-phosphorylation, effectively acting as a competitive antagonist for Tie2.
Interestingly, Ang2 is able to elicit agonistic effects when driven outside of its natural
physiological state, such as at high concentrations [270, 271], after prolonged exposure, as
engineered high-order oligomers [227], or in non-endothelial cells transfected with Tie2
[272], supporting the hypothesis that the antagonistic role of Ang2 in its physiological state is
at least in part mediated by its natural tendency to form lower order oligomers.
Ang2 is expressed by endothelial cells [232] and perivascular smooth muscle [237] at
sites of active angiogenesis during the vessel destabilization process [230, 272, 273]. In
quiescent vessels, Ang2 is stored in intracellular Weibel-Palade bodies in endothelial cells
along with von Willebrand factor, which is involved in hemostasis [274]. Stored Ang2 has a
half-life of 18 hours, but can be secreted within minutes of stimulation [274]. In adults, Ang2
secretion is induced by hypoxia [230, 237, 275], and cytokines such as Hypoxia-Inducible
Factor (HIF) 1α [276], VEGF [277], Basic Fibroblast Growth Factor (bFGF) [237], and
inflammatory mediators Tumour Necrosis Factor (TNF) α and NFκB [278], or vasoactive
molecule thrombin [274, 279]. As the antagonist of Ang1, Ang2 induces the destabilization
28
of vessel integrity [277, 280-282], thus increasing BSCB permeability, and thereby allowing
vessels to undergo remodelling [283, 284]. Destabilization of existing vessels increases
endothelial plasticity and is a primary prerequisite to vascular remodelling. The result of
these permeability changes on angiogenesis also depend on the local cytokine milieu. In the
presence of growth factors such as VEGF, Ang2 induces angiogenic sprouting [281, 285,
286]; while in the absence of VEGF, Ang2 destabilization leads to vessel regression [281,
286-288]. Ang2 induced increase in BSCB permeability primes the endothelium and
mediates the escalation of the inflammation response by allowing the passage of
inflammatory cells through CNS vessels [287, 289]. By loosening endothelial cell junctions,
Ang2 facilitates the migration of inflammatory cells from the bloodstream into the CNS [278,
289].
1.5.3
Angiogenin
Angiogenin is a 123 amino acid, 14 kDa protein that induces angiogenesis and
neovascularisation [290-293]. It was the first reported tumour-derived angiogenic protein,
characterized by Fett and Strydom in 1985 [290, 294]. Angiogenin shares 65% homology to
bovine pancreatic RNase A [294, 295], and although it has surprisingly low ribonucleolytic
activity [296], the ribonucleolytic site on Angiogenin appears to be essential for its
angiogenic actions [291, 297-301]. Angiogenin is predominately expressed and released by
endothelial cells, but it is also widely expressed by a variety of anchorage-dependent
proliferating cells including aortic smooth muscle cells, fibroblasts, and various tumour cells
[302]. Angiogenin expression is induced by HIF1α under cellular stress or hypoxic
conditions [303-305].
29
A 170 kDa receptor has been identified as the Angiogenin receptor [306]. Upon
binding, Angiogenin is translocated to the nucleus where it regulates genes controlling the
proliferation of endothelial cells by activation of ERK [307], Akt [308], and the SAPK/JNK
pathways [309]. Angiogenin has also been reported to interact with 42 kDa smooth muscle
type α actin [310, 311]. This interaction induces the formation of Angiogenin-actin
complexes, which drive the degradation the extracellular matrix and basement membrane of
blood vessels in order to promote the migration of vascular components [297, 312].
Aside from its prominent role in promoting tumour angiogenesis [290, 292, 302, 313,
314], Angiogenin is well characterized in the pathogenesis of ALS. Genetic mutations in
Angiogenin have been linked to both familial and sporadic ALS [220, 221, 315]. These ALSassociated mutants appear to have reduced or abolished survival-promoting activity, leading
to the degeneration of motor neurons, an apparent symptom in the pathogenesis of ALS [222,
303, 316].
1.5.4
The Expression and Role of Angiogenic Cues outside the Vascular System
Outside the vascular system, Ang1, Ang2 and Tie2 expression have been reported in
a variety of cells in the nervous system. Ang1 expression has been reported in immature cells
of the subventricular zone (SVZ) [317], motor neurons [318], and astrocytes [319, 320]
during development, while Tie2 expression has been detected in the dorsal root ganglion
[321], the SVZ [322, 323], neural progenitor cells [324, 325], neurons [321, 326] and
Schwann cells of the PNS [266, 327, 328].
Ang1 and Ang2 both have neurotrophic effects, inhibiting apoptosis, promoting
proliferation, differentiation, and maturation of neural progenitor cells (NPC). Ang1 has been
30
reported to prevent apoptosis in oxygen or glucose-deprived NPC [329] and neurons [326].
Ang1 has also been implicated in the proliferation of NPC [322, 324, 330], their
differentiation into neurons [317, 321, 331], neurite outgrowth [321, 322, 331-333], as well
as their organization into a functional neuronal network [334]; while Ang2 has been
implicated in neurogenesis in the SVZ [330, 335].
The convergence of the vascular and nervous systems is yet again apparent with
regards to the Angiopoietin proteins. In context of this thesis work, vascular disruption,
ischemia, and BSCB permeability all contribute to secondary injury after SCI. Therefore,
understanding angiogenesis and manipulating it therapeutically may potentially restore
perfusion, reduce ischemic insults, reconstitute the BSCB and ultimately limit secondary
injury.
1.5.5
Angiogenic Proteins as Treatment after Spinal Cord Injury
Indeed, Ang1 as a treatment resulted in improved functional and histological
outcomes following experimental SCI [336, 337]. Ang1 with the integrin peptide C16 was
shown to rescue vasculature at the injury epicentre [336]. There was also an increased
amount of spared white matter at both 7 and 42 days post-injury [336], although no further
improvement was observed at 42 than 7 days. Decreased inflammation was observed as early
as 24 hours post-injury, along with behavioural improvements from 7 to 42 days post-injury
(Basso Mouse Scale [338]) [336].This suggests that targeting early vascular mechanisms can
indeed result in long-term functional improvements by rescuing vasculature and reducing
some aspects of inflammation very soon after SCI. This study is of particular clinical
31
importance, because treatment did not begin until 4 hours post-injury, which is a clinically
realistic timeframe for a neuroprotective intervention in human SCI [336].
In another study, the combination of Ang1 and VEGF treatment by Adeno-Associated
Virus (AAV) transfection in rats immediately after SCI decreased lesion volume and
promoted vascular stability [337]. This treatment decreased edema, demyelination, and
BSCB permeability, resulting in improved open-field locomotor function at 56 days postinjury (Basso-Beattie-Bresnahan locomotor rating scale [339]) [337]. Interestingly, the
combination of Ang1 and other angiogenic molecules such as C16 or VEGF both resulted in
synergistic functional outcome compared to each individual treatment, suggesting that in
addition to the roles that Ang1 plays in the formation of stable vessel, the regulation of
BSCB permeability, and promotion of cell survival, further benefits may result from the
administration of these exogenous angiogenic agents to promote angiogenesis and maintain
vascular perfusion to the injury penumbra and limit secondary injury after SCI.
1.6
Research Objectives
The previous sections outlining the vascular changes that occur after SCI highlights
the fundamental need to understand the pathophysiology of the secondary spinal cord
damage that rapidly follows the primary injury. It has become evident from a vast body of
literature in rodent models of SCI that the processes that mediate secondary injury are
extremely complex, and the precise interactions of angiogenic signalling in the spinal cord
remain elusive.
The general objective of this research is to investigate the damage to local vasculature
and the endogenous angiogenic remodelling response after acute SCI. Specifically, this thesis
32
will focus on one aspect of secondary injury after SCI: the vascular/angiogenic changes that
occur after acute SCI. Based on the reported endothelial damage and vascular leakage after
acute SCI, I hypothesize that Ang1 levels will decrease while Ang2 levels will increase after
SCI. To represent these changes, the endogenous expression of Ang1, Ang2, and Angiogenin
in CSF after acute human SCI, and Ang1 and Ang2 in rat spinal cord will be characterized.
These will be addressed in two studies: one focused on the human condition, and the other
based in a small rodent model of SCI.
Study #1
Objective: To characterize the endogenous protein changes in Angiopoietin-1, Angiopoietin2, and Angiogenin in cerebrospinal fluid after acute human spinal cord injury.
Rationale: Angiogenic proteins play a substantial role in promoting survival of endothelial
cells and thus regulating perfusion to the injury penumbra and permeability of the bloodspinal cord-barrier, factors which have been implicated in the propagation of secondary
damage after spinal cord injury.
This study is presented in Chapter 2.
Study #2
Objective: To characterize the endogenous protein changes of Angiopoietin-1 and
Angiopoietin-2 in the spinal cord of rats after acute spinal cord injury.
33
Rationale: Having examined the expression of angiogenic proteins in human spinal cord
injury patients, this study will examine if the trends observed in the previous study are
similar in firstly, spinal cord tissue, and secondly, in an animal model of spinal cord injury.
This study is presented in Chapter 3.
34
Chapter 2: Changes in Angiogenic Proteins after Acute Human Spinal
Cord Injury
2.1
Introduction
The sequelae of events following the initial (primary) SCI, which constitute the
secondary injury cascade, have been well documented. Nemecek used the term ‘autodestruction’ of the spinal cord following SCI to describe the continual progression of cell
death after SCI [340]. Damage to vasculature both as a result of the initial mechanical
impact; and subsequently due to progressive cell death, is apparent, and is central to the
evolution of the expansion of the damage into previously unaffected injury penumbra.
Vascular dysfunction after SCI propels secondary injury via ischemic and hypotensive
mechanisms, restricting metabolic supply; but also via the propagation of inflammation, and
the accumulation of neurotoxic and cytotoxic molecules into the injury penumbra as the
BSCB breaks down. This ultimately leads to the deterioration of neurological deficits over
the acute and sub-acute phase post-injury.
Much of the current knowledge regarding the vascular disturbances after SCI has
revolved around what has been studied and reported in animal models of SCI. Studying the
biology of human SCI is obviously considerably more difficult than in animal models given
that cord specimens can only be obtained post-mortem. In the current study, repeated CSF
samplings were used as a biological proxy for biochemical changes in the spinal cord, to
illustrate the temporal progression of the vascular pathology of SCI. Ang1, Ang2, and
Angiogenin are all secreted proteins [223, 230, 235, 274, 305, 311], which activate receptors
on endothelial cells. Therefore, it can be inferred that molecules of these angiogenic proteins,
once released, will reach the CSF circulation to allow for detection.
35
In this current study, temporal changes in three angiogenic proteins: Ang1, Ang2, and
Angiogenin were examined following acute human SCI. Protein levels were determined in
both CSF and serum samples of SCI patients and non-SCI controls to characterize the
patterns of expression of these proteins and how their levels might be influenced by injury
severity and/or hold implications for neurological recovery after SCI.
2.2
2.2.1
Materials and Methods
Patient Enrollment and Clinical Evaluation
Patients from a single level 1 regional trauma institution were enrolled in a clinical
trial in which lumbar intrathecal catheters were inserted to measure CSF pressure and obtain
CSF samples [341]. The patients provided informed consent to participate in the study in
which an intrathecal drain was installed and left in situ for 3 to 5 days.
The clinical trial protocol was approved by the university human ethics committee,
and was registered on ClinicalTrials.gov (ID: NCT00135278). Inclusion criteria for
enrolment included: 1) SCI between cervical (C) 3 and thoracic (T) 11 inclusive; 2) ASIA
Impairment Scale (AIS) A – motor and sensory complete SCI, B – motor complete, sensory
incomplete SCI, or C – motor and sensory incomplete SCI upon presentation; 3) presentation
within 48 hours of injury; and 4) ability to provide a valid and reliable baseline neurological
exam. Patients with concomitant head injuries, major trauma to the chest, pelvis, or
extremities that required invasive intervention and those who were too sedated or intoxicated
to give a valid neurologic examination were excluded.
Upon presentation, patients were evaluated by a clinical research nurse and a
neurological examination was performed to assign a baseline AIS score. Long-term outcome
36
was measured at 6-month and 1-year post-injury with parameters used in the initial baseline
neurological testing including AIS, ASIA motor score, and the last normal sensory level. To
obtain control CSF from the “non-SCI” condition, a direct lumbar dural puncture was
performed in individuals undergoing lumbar spine surgery.
2.2.2
Sample Collection and Processing
Intrathecal catheters (PERIFIX® Custom Epidural Anaesthesia Kit; B. Braun
Medical Inc., Bethelehem, PA, USA) were inserted at L2/3 or L3/4 and CSF was collected
every 6 to 8 hours for 3 to 5 days using a strict aseptic technique. For non-SCI controls
undergoing lumbar spine surgery, a sample of CSF was obtained via needle puncture of the
dura at the end of their surgery. Samples were immediately centrifuged at 1000 rcf for 10
minutes. The supernatant was aliquoted, snap frozen in an ethanol and dry ice bath, and
stored at -80°C until analysis. Blood samples were drawn in both SCI patients and non-SCI
controls at the same times that CSF samples were collected. The blood samples were left to
clot at room temperature for 15 minutes, and then centrifuged at 10,000 rcf for 5 minutes.
The serum was aliquoted, frozen and stored at -80°C until analysis.
2.2.3
Molecular Analysis
The CSF and serum samples were analyzed using standard quantitative sandwich
ELISA kits for Ang1 (Quantikine® Human Angiopoietin-1) and Ang2 (Quantikine® Human
Angiopoietin-2), and a microparticle-based multiplex ELISA kit for Angiogenin
(Fluorokine® MAP Human Angiogenesis Base Kit A and Angiogenin bead set). All kits
were manufactured by R&D Systems Inc., Minneapolis, MN, USA. For SCI patients, up to
37
15 CSF and serum samples taken between 8 and 120 hours post-injury were analyzed. A
single baseline sample was analyzed for non-SCI controls. All samples were run in duplicate.
2.2.4
Statistical Analysis
Statistical analysis was performed using SPSS Statistics 18.0 software. Normal
distribution in the data was tested using the Shapiro-Wilk test, and equality of variances
between groups was tested using the Levene test. Because the sample population did not
show normal distribution, non-parametric statistical tests were used for comparing group
values. The Mann-Whitney U-test was used to compare values between SCI patients and
non-SCI controls at each time point. The Kruskal-Wallis H-test was used to compare protein
levels with baseline AIS classification. The Friedman test was used to examine temporal
changes in individual patients. Spearman’s correlation was used to investigate relationships
between protein levels and outcome parameters at 6-month or 1-year post-injury. Data is
presented as group (SCI or non-SCI) means with the standard error of the mean (SEM).
Statistical significance is reported at p < 0.05.
2.3
Results
Fifteen SCI patients equally divided amongst injury severity at baseline (five AIS A,
five AIS B, and five AIS C) were analyzed (Table 2.1). Twelve individuals suffered cervical
SCI, while three suffered thoracic SCI. The average age was 41.7 years, with 13 males and 2
females. Five patients were injured by motor vehicle accidents, five during sporting
activities, four from falls, and one from a direct blow to the back of the head (Table 2.1). The
CSF and serum of these 15 patients were compared against 8 non-SCI control subjects. This
population averaged 60.1 years in age, and included 4 males and 4 females (Table 2.2).
38
Table 2.1
Demographics of SCI patients enrolled in the current study.
* denotes 6-month outcome data where 1-year outcome was unknown or not yet performed at the time this thesis was prepared.
UNK is unknown, where follow-up neurological testing was not performed or not yet performed at the time this thesis was prepared.
Subject
Age/
Sex
Injury
level
Mechanism of
injury
SCI 1
SCI 2
SCI 3
SCI 4
SCI 5
SCI 6
SCI 7
SCI 8
SCI 9
42/M
64/F
66/M
60/M
46/M
20/M
39/M
54/M
19/F
C6-7
C5-6
C4-5
C4-5
C4-5-6
C6-7
C3-4
C5-6
T10
SCI 10
38/M
C5
SCI 11
SCI 12
SCI 13
SCI 14
SCI 15
22/M
25/M
51/M
55/M
25/M
L1
C7
T10
C6-7
C5
Transport
Sports
Fall
Fall
Transport
Transport
Sports
Transport
Fall
Struck on back of
head
Transport
Sports
Fall
Sports
Sports
Upon presentation
Last
ASIA
normal
AIS
motor
sensory
score
level
6-month (*) or 1-year follow-up
Last
ASIA
normal
AIS
motor
sensory
score
level
B
C
C
B
C
B
C
C
A
19
41
20
6
5
30
33
36
50
C5
C5
T9
C5
C2
C6
C3
C4
T9
B
D
C*
D
C*
C*
D
D*
A
27
98
49*
80
71
86*
83
65*
50
C6
C2
C4*
C4
C5
T6*
C4*
C5*
T7
B
17
C5
D*
15*
C6*
A
A
A
A
B
60
43
50
27
13
C2
C7
T11
C6
C4
A*
C
A*
UNK
B*
66*
58
50*
UNK
UNK
L2*
C7*
T11*
UNK
C4*
39
Table 2.2
Demographics of non-SCI subjects enrolled in the current study.
Subject
CTRL 1
CTRL 2
CTRL 3
CTRL 4
CTRL 5
CTRL 6
CTRL 7
CTRL 8
Age/Sex
49/M
85/F
52/F
68/F
45/M
61/F
62/M
59/M
Diagnosis
L4-5 recurrent disc herniation
L2-3, 3-4, 4-5, 5-S1 stenosis
Right S1 radiclopathy
Degenerative L3-4 spondylolisthesis
L5-S1 disc herniation
L5-6 disc herniation
L5-S1 degenerative disc disease with neural foraminal stenosis
L3-4 spinal stenosis; L4-5 degenerative spondylolisthesis
40
The concentrations of Ang1, Ang2, and Angiogenin were evaluated at 12-hour
intervals from 24 hours to 120 hours (5 days) post-injury. The values within each group did
not display normal distribution (Shapiro-Wilk test), and variances between groups were
unequal (Levene test), hence non-parametric statistical tests were used for all comparisons.
No significant differences were found in Ang1, Ang2, or Angiogenin protein concentrations
were found between AIS A, B, or C patients at any time point post-injury (including
baseline); therefore, data from all injury severities (AIS A, B, and C) were pooled to compare
the SCI condition against non-SCI controls. For non-SCI controls, only a single CSF and
serum sample was obtained. It was assumed that the concentrations would remain largely
unchanged over time in these individuals. It has been established that protein concentrations
in CSF are much more variable between individuals than within individuals [342].
For Ang1, both the CSF and serum levels of Ang1 at the earliest time point analyzed,
around 24 hours post-injury, was higher in SCI patients than non-SCI controls (Figure 2.1,
Table 2.3). The mean Ang1 concentration in CSF in SCI patients at this time was 92% higher
than that of non-SCI controls; with SCI mean at 83.48 ± 18.86 pg/ml and non-SCI controls at
43.38 ± 4.63 pg/ml (p = 0.028, Mann-Whitney U-test). In the serum, the mean value for SCI
patients at 24 hours post-injury was 5124.12 ± 524.79 pg/ml, and in non-SCI controls
2903.96 ± 325.68 pg/ml (p = 0.025, Mann-Whitney U-test). These elevations in Ang1
diminished over the subsequent 12 hours, and by 36 hours post-injury, SCI and non-SCI
control levels were no longer significantly different in CSF or serum. There were also no
statistically significant correlations between Ang1 levels and 6-month or 1-year neurologic
outcomes (Spearman’s rank correlation coefficient).
41
Figure 2.1 Mean Ang1 protein levels in CSF and serum after acute human SCI.
Mean CSF value for SCI patients at 24 hours post-injury was 83.48 ± 18.86 pg/ml whereas mean value for
non-SCI controls was 43.38 ± 4.63 pg/ml (p = 0.028), representing a 92% increase in Ang1 protein levels
in CSF. Mean serum value at 24 hours post-injury for SCI patients was 5124.12 ± 524.79 pg/ml, and
mean value for non-SCI controls was 2903.96 ± 325.68 pg/ml (p = 0.0125), representing a 76% increase.
Blue lines (circles) represent mean CSF values and red lines (triangles) represent mean serum values. SCI
patients are represented with solid lines and non-SCI controls are represented with dotted lines. The
dotted lines show a single sampling from non-SCI controls, and does not represent the temporal changes
through time. It is simply a tool to ease the comparison to SCI values. Data presented as mean ± SEM. *
denotes p < 0.05, Mann-Whitney U-test.
42
In contrast to the early peak in Ang1, a delayed and sustained increase in CSF Ang2
protein expression was observed (Figure 2.2, Table 2.4). Increased Ang2 was observed in
SCI patient CSF from 36 hours post-injury and stay elevated until the end of the study
period. A 2.3-fold increase was observed in Ang2 expression at 120 hours post-injury, with
levels still apparently rising at this time point (no further CSF samples were collected after
120 hours, as established by the clinical trial protocol). Ang2 mean value for SCI patients at
120 hours post-injury was 815.66 ± 197.15 pg/ml whereas non-SCI control mean at 354.45 ±
32.07 pg/ml (p = 0.048, Mann-Whitney U-test). The maximal difference in serum levels is
observed at 48 hours post-injury, with SCI patient mean at 3071.93 ± 520.07 pg/ml, and nonSCI control mean at 1628.05 ± 210.78 pg/ml. The differences between SCI and non-SCI
values were statistically significant in CSF from 36 hours until the end of the study, and in
serum between 48 and 60 hours post-injury (p = 0.033, Mann-Whitney U-test). Again, no
significant correlations were found between Ang2 values and the neurologic outcome at 6month or 1-year post-injury (Spearman’s rank correlation coefficient).
43
Figure 2.2 Mean Ang2 protein levels in CSF and serum after acute human SCI.
SCI patients showed significantly higher Ang2 values from 36 hours post-injury until the end of the study
period at 120 hours post-injury in the CSF. Serum samples from SCI patients were also significantly
higher than non-SCI controls between 48 and 60 hours post-injury. The level in CSF at 120 hours postinjury was 815.66 ± 197.15 pg/ml (p =0.048) in SCI patients and non-SCI control value at 354.45 ± 32.07
pg/ml. This represents a 130% increase in Ang2 levels in CSF. Serum levels showed the greatest increase
at 48 hours, with mean SCI value at 3071.93 ± 520.07 pg/ml compared to non-SCI mean at 1628.05 ±
210.78 pg/ml (p = 0.033), representing a 89% increase.
Blue lines (circles) represent mean CSF values and red lines (triangles) represent mean serum values. SCI
patients are represented with solid lines and non-SCI controls are represented with dotted lines. The
dotted lines show a single sampling from non-SCI controls, and does not represent the temporal changes
through time. It is simply a tool to ease the comparison to SCI values. Data presented as mean ± SEM. *
denotes p < 0.05, Mann-Whitney U-test.
44
For Angiogenin protein levels in the CSF, the concentration in SCI patients appeared
to decrease starting around 36 hours post-injury, and between 72 and 84 hours post-injury,
levels were significantly lower than in non-SCI controls (Figure 2.3, Table 2.5). Maximal
decrease was observed at 84 hours post-injury with mean value for SCI was only 83% that of
non-SCI controls, at 7.79 ± 1.05 ng/ml and mean value for non-SCI at 9.3 ± 0.6 ng/ml (p =
0.033, Mann-Whitney U-test). In the serum, there were no changes until 60 hours post-injury,
with an increasing trend from 60 to 120 hours post-injury. Serum levels were significantly
higher in SCI patients at 120 hours post-injury, with a mean of 2.18 ± 0.41 ng/ml compared
to non-SCI mean at 1.63 ± 0.21 ng/ml (p = 0.025, Mann-Whitney U-test). There were also no
significant correlations were found between Angiogenin values and the neurologic outcome
at 6-month or 1-year post-injury (Spearman’s rank correlation coefficient).
45
Figure 2.3 Mean Angiogenin protein levels in CSF and serum after acute human SCI.
SCI patients showed significantly lower Angiogenin values in CSF between 72 and 84 hours post-injury.
No significant differences were observed between SCI patients and non-SCI controls in serum samples.
Mean CSF value in SCI patients at 84 hours post-injury was 7.80 ± 1.05 ng/ml and mean for non-SCI
controls was 9.3 ± 0.60 ng/ml (p = 0.033). Serum levels at 120 hours was 2.18 ± 0.41 ng/ml (p = 0.025).
Blue lines (circles) represent mean CSF values and red lines (triangles) represent mean serum values. SCI
patients are represented with solid lines and non-SCI controls are represented with dotted lines. The
dotted lines show a single sampling from non-SCI controls, and does not represent the temporal changes
through time. It is simply a tool to ease the comparison to SCI values. Data presented as mean ± SEM. *
denotes p < 0.05, Mann-Whitney U-test.
46
Table 2.3
Expression of Ang1 expression in CSF after acute human SCI.
Data shown as mean (range) in pg/ml.
Non-SCI
43.38
(29.11 –
66.01)
Table 2.4
24
83.48
(31.72 –
313.40)
36
67.04
(21.11 –
180.78)
48
50.63
(28.58 –
120.35)
Hours post-injury
60
72
44.56
42.27
(18.63 –
(17.45 –
97.59)
75.80)
84
43.94
(19.89 –
77.16)
96
46.62
(21.09 –
78.47)
120
45.89
(13.06 –
66.25)
96
772.91
(183.33 –
2983.04)
120
815.66
(191.55 –
3182.39)
96
8.06
(3.32 –
16.84)
120
8.10
(3.30 –
19.12)
Expression of Ang2 expression in CSF after acute human SCI.
Data shown as mean (range) in pg/ml.
Non-SCI
354.45
(260.51 –
514.76)
Table 2.5
24
508.37
(244.91 –
119.49)
36
640.11
(252.15 –
2014.76)
48
722.58
(237.11 –
1843.88)
Hours post-injury
60
72
645.44
618.17
(223.97 –
(212.02 –
1610.09)
1468.79)
84
745.66
(218.62 –
2783.68)
Expression of Angiogenin expression in CSF after acute human SCI.
Data shown as mean (range) in ng/ml.
Non-SCI
9.3
(7.04 –
12.17)
24
10.50
(5.84 –
17.72)
36
11.18
(3.86 –
30.76)
48
8.80
(3.28 –
19.17)
Hours post-injury
60
72
7.74
7.36
(3.31 –
(3.37 –
17.77)
17.12)
84
7.80
(3.34 –
18.53)
47
2.4
Discussion
This study has established a method to illustrate temporal changes in three angiogenic
proteins: Ang1, Ang2, and Angiogenin after acute human SCI. In summary, Ang1 in CSF
showed high levels of protein expression early after SCI, thereafter decreasing to non-SCI
control values at 36 hours post-injury, and maintaining this level throughout the duration of
the study. Ang2 protein in the CSF showed the opposite pattern as Ang1 (Figure 2.4). Ang2
was up-regulated at 36 hours post-injury, continuing to rise until 120 hours post-injury.
Angiogenin protein did not show any remarkable changes in the CSF, but did exhibit a late
increase at 120 hours post-injury in the serum.
Figure 2.4
A comparison of Ang1 and Ang2 protein expression in CSF after acute human SCI.
Blue lines (closed circles) represent mean Ang1 values in CSF and green lines (open circles) represent
mean Ang2 values in CSF. SCI patients are represented with solid lines and non-SCI controls are
represented with dotted lines. The dotted lines show a single sampling from non-SCI controls, and does
not represent the temporal changes through time. It is simply a tool to ease the comparison to SCI values.
Data presented as mean ± SEM. * denotes p < 0.05, Mann-Whitney U-test.
48
The absence of a sustained increase in Ang1 levels within the CSF in the current
study supports the contention of investigators such as Han [336] and Herrera [337] who
administered Ang1 in the hopes of increasing angiogenesis and restoring the integrity of the
BSCB after acute SCI. These authors showed that Ang1 alone or in combination with other
angiogenic factors such as VEGF, resulted in functional improvements after experimental
SCI [336, 337]. A previous attempt to measure VEGF levels was unsuccessful (below
detectable limits of the similar biochemical assay) in the CSF of the current series of SCI
patients, in a separate attempt to investigate the biochemical changes in CSF after acute
human SCI [19].
A decrease in the expression of Ang1 messenger ribonucleic acid (mRNA) has been
reported after acute SCI in rats from 6 hours to 2 weeks post-injury [343]. Increased Ang1
expression could limit the spread of secondary damage by tightening paracellular interactions
between endothelial cells and mural cells to close paracellular junctions in vessel walls, and
preserve the integrity of the BSCB. Compromised BSCB after SCI promotes the invasion of
inflammatory cells and other toxic blood products into the injury penumbra [152, 156, 190].
Ang1 activity early after SCI could eliminate the extra-vascular space which has been
reported to become a conduit for inflammatory cells invading the site of injury [199].
Furthermore, Ang1 also promotes survival of endothelial cells [238-241]. By preserving
distal circulation in the overlapping vascular networks of the spinal cord, adequate perfusion
to the injury penumbra could be maintained.
In the current study, a significantly higher level of Ang2 was detected in the CSF. No
significant correlation was found between these levels or their long-term (6-month or 1-year)
functional outcome examined using the ASIA neurological test, and how the outcome (if
49
any) of elevated Ang2 levels in CSF after SCI affects the pathophysiology of SCI or the
eventual neurological outcome remains elusive. However, it can be postulated that this
prolonged up-regulation of Ang2 expression could exacerbate secondary injury by
destabilizing endothelial junctions to increase BSCB permeability. This corresponds to the
delayed angiogenic response after SCI that has been reported in several studies, ranging from
3 to 7 days post-injury [147, 156, 164, 188, 190, 199]. However, no further neurological
changes were correlated to the new angiogenic status in these studies, adding evidence to
support that although endogenous reparative response of the damaged tissue and vasculature
is observed after acute SCI, these neovessels fail to integrate into a functional NVU. It is
likely that the increase in BSCB permeability during the early stages of injury, in addition to
the lack of a continued robust Ang1 response to promote vascular stability, contributes to the
exacerbation of secondary injury. Angiogenesis and the maintenance of BSCB permeability
after acute traumatic SCI is not only a neuroprotective strategy – to preserve remaining
neurons and glia and prevent further cell death by ischemia; but blood vessels also provide
trophic support [217-219] and a scaffold for both endogenous and potential regeneration
strategies at later time points after SCI [344-347].
However, along with stimulating angiogenesis, this prominent increase in Ang2 after
SCI may allow the passage of deleterious inflammatory cells and cytokines, as well as
cytotoxic molecules into the injury penumbra, leading to further cell death. A marked
inflammatory response has been established from 3 to 7 days post-injury [156, 190, 198],
which coincides with revascularization of the injury epicentre [151, 154-156]. The time
course of BSCB repair also closely parallels that of the appearance of the glial scar [155]. It
has recently been reported that a subpopulation of pericytes, perhaps those which proliferate
50
during the first angiogenic stage, but are not integrated into a functional NVU, contribute
significantly to the glial scar. These pericytes migrate out of the vessel wall, transdifferentiate to become fibroblast-like, move into the lesion core and are responsible for a
majority of the extracellular deposition of the glial scar sealing the injury epicentre [115].
Interestingly, there were no remarkable changes observed in Angiogenin expression
in CSF after acute SCI in the current study. This suggests that the angiogenic changes that
occur in the acute phase post-injury are not driven by the same mechanism as the
angiogenesis in many carcinogenic tumours that has been reported to be associated with
Angiogenin [290, 313, 314]. However, an increase in Angiogenin in serum at 120 hours postinjury was observed. This could be indicative of an angiogenic response to systemic injuries,
as many SCI patients are admitted with multiple trauma. It is also possible that the large
variations observed in the current study, particularly in Angiogenin protein levels, may have
masked any potential differences between SCI patients and non-SCI controls.
The temporal changes examined in this study represent the changes of all SCI patients
compared to all non-SCI controls in the current study population. However, it has been
reported that there is substantial variation in inter-individual protein expression levels in
CSF, even in healthy individuals [342]. Considerable variation in protein expression was also
observed in the current series of SCI patients and uninjured controls. Certain patterns of
change present after SCI in our sample population could potentially have been masked by
this great inter-individual variation, as it would be logistically impossible to acquire a nonSCI baseline measure for each SCI patient enrolled in this study to use as a comparison for
their post-injury expression values. However, a majority of the values recorded for our non-
51
SCI controls reflect the values of controls which have previously been reported [313, 314,
348-363] (Table 2.6).
In the current study, all protein levels in serum were considerably higher, by orders of
magnitude, than in CSF. However, the serum and CSF concentrations did not appear to be
changing in parallel, which provides evidence to support that the changes seen in the CSF are
indeed local CNS changes, and not a spill-over from serum expression due to systematic
injuries. This is evident in the changes seen in the balance between Ang1 and Ang2
expression. While serum Ang2 levels were much lower than that of serum Ang1, CSF Ang2
is more than one order of magnitude higher than CSF Ang1. This is not unexpected, as Ang1
is constitutively expressed in the maintenance of quiescent adult vessels, while Ang2 is only
found at sites of active angiogenesis. Given the context of CNS trauma, and the antagonistic
role of Ang2 against Ang1, this suggests that the observed increase in CSF Ang2 may indeed
an active up-regulation in the expression and/or secretion of Ang2 in the CNS due to SCI,
and may indicate the destabilization of local vasculature and breakdown of BSCB after SCI.
2.5
Conclusions
This chapter presents novel findings on the expression of three angiogenic proteins:
Ang1, Ang2 and Angiogenin in CSF and serum after acute human SCI. The delayed and
sustained Ang2 protein levels in CSF in addition to the lack of Ang1 up-regulation in SCI
patients in the current study may suggest a possible link between Ang2 up-regulation, the
increased permeability of the BSCB [190], and possible further deterioration of secondary
pathologies after SCI. These include but are not limited to the invasion of immune cells, the
loss of ionic and metabolic homeostasis, infiltration of cytotoxic or neurotoxic molecules
52
(including blood products, reactive oxygen species, neurotransmitters, et cetera) into the
CNS to further cell death and propagate neurological deterioration. This is compounded by
ischemia and the induction of metabolic stress induced by vascular dysfunction after SCI.
The intimate relationship of the NVU and its pathophysiology remains an important focus as
a neuroprotective/neuro-regenerative strategy for SCI and other CNS disorders alike.
53
Table 2.6
Summary of serum and CSF Ang1 values reported in the current study and in literature.
Data shown as mean ± SD and (range); or median and [IQR]. Control values are shown in italics.
Author
Ng
Joshi
Reed
Choe
Han
Karapinar
Anagnostopoulos
Study
Current study
Clin Biochem.
2011.
Kidney
International.
2011.
Joint Bone
Spine. 2010.
Hypertens
Pregnancy.
2010.
Heart and
Vessels. 2010.
Br J Haematol.
2007.
Population
SCI (n=15)
Ctrl (n=8)
Multiple myeloma (n=62)
Ctrl (n=50)
Autosomal dominant
polycystic kidney disease
(n=71)
Bencet’s disease (n=59)
Ctrl (n=65)
Pre-eclampsia (n=16)
Ctrl (n=29)
Hypertension (n=49)
Ctrl (n=21)
Waldenstrom’s
macroglobulinemia
(n=56)
Ctrl (n=30)
Serum (ng/ml)
CSF (pg/ml)
At 24 hours post-injury:
At 24 hours post-injury:
5124.12 ± 523.79
2903.96 ± 325.68
36.28 (19.8 – 44.0)
37.05 (35.7 – 39.2)
83.48 ± 18.86
43.38 ± 4.63
35.52 ± 21.03
284.5 ± 101.2
237.1 ± 76.4
Plasma
12.65 (1.27 – 17.5)
10.35 (1.43 – 31.89)
26.95 ± 11.63
43.34 ± 9.77
18.4 (1.7 – 107.5)
23.2 (0.1 – 45.9)
54
Table 2.7
Summary of serum and CSF Ang2 values reported in the current study and in literature.
Data shown as mean ± SD and (range); or median and [IQR]. Control values are shown in italics.
Author
Study
Population
Ng
Current study
SCI (n=15)
Ctrl (n=8)
Joshi
Clin Biochem.
2011.
Multiple myeloma (n=62)
Ctrl (n=50)
Kidney
International.
2011.
Hypertens
Pregnancy.
2010.
Clin Cancer
Res. 2009.
Amyotroph
Lateral Scler.
2009.
Autosomal dominant
polycystic kidney disease
(n=71)
Reed
Han
Helfrich
Moreau
Anagnostopoulos
Br J Haematol.
2007.
Pre-eclampsia (n=16)
Ctrl (n=29)
Melanoma (n=98)
Ctrl (n=82)
Serum (ng/ml)
CSF (pg/ml)
At 120 hours post-injury:
At 120 hours post-injury:
2184.5 ± 406.55
1628.05 ± 210.78
4.45 (2.1 – 13.25)
1.67 (0.25 – 3.45)
815.66 ± 197.15
354.45 ± 32.07
2.35 ± 0.96
Plasma
11.2 (2.3 – 21.9)
3.9 (1.4 – 14.7)
2.03 [1.71 – 3.28]
1.24 [0.93 – 1.57]
ALS (n=40)
Ctrl (n=40)
Waldenstrom’s
macroglobulinemia
(n=56)
Ctrl (n=30)
86.75 [67 – 132]
82.5 [30 – 147]
2.6 (1.0 – 11.3)
1.4 (0.6 – 5.1)
55
Table 2.8
Summary of serum and CSF Angiogenin values reported in the current study and in literature.
Data shown as mean ± SD and (range); or median and [IQR]. Control values are shown in italics.
Author
Ng
Moreau
Ilzecka
Study
Current study
Amyotroph
Lateral Scler.
2009.
Acta Clin
Croat. 2008.
Patel
Ann Med.
2008.
Anagnostopoulos
Br J Haematol.
2007.
Huang
Eur Nerol.
2007.
Siebert
Diabetes Care.
2007.
Cronin
Kim
Neurology.
2006.
Leukemia and
Lymphoma.
2005.
Population
SCI (n=15)
Ctrl (n=8)
Serum (ng/ml)
CSF (ng/ml)
At 84 hours post-injury:
At 84 hours post-injury:
379.23 ± 54.84
279.76 ± 47.71
7.80 ± 1.05
9.3 ± 0.6
ALS (n=40)
Ctrl (n=40)
288.0 [267 – 307]
282.5 [244 – 326]
ALS (n=20)
Ctrl (n=15)
Chronic heart failure
(n=109)
Ctrl (n=112)
Waldenstrom’s
macroglobulinamia
(n=56)
Ctrl (n=30)
Acute cerebral infarction
(n=30)
Ctrl (n=20)
Diabetes mellitus type 2
(n=43)
Ctrl (n=43)
ALS (n=79)
Ctrl (n=72)
0.328 (0.208 – 0.45)
0.286 (0.153 – 0.483)
Leukemia (n=43)
Ctrl (n=18)
466 [314 – 739]
310 [264 – 376]
398.1 (147.4 – 1180.6)
226.9 (145.8 – 398.7)
At 48 hours:
415.1 ± 76.8
334.9 ± 93.9
319.7 ± 107.04
550.54 ± 187.99
396.7 ± 120.9
334.6 ± 106
277.6 (145.9 – 533.7)
226 (68 – 349.8)
56
Author
Molica
Study
Eur J
Haematol.
2004.
Hisai
Clin Cancer
Res. 2003.
Verstovsek
Br J Haematol.
2001.
Miyake
Cancer. 1999.
Population
Serum (ng/ml)
Leukemia (n=77)
Ctrl (n=15)
295 (74 – 1700)
264 (29 – 1835)
Hepatocellular carcinoma
(n=39)
Ctrl (n=31)
Leukemia /
myelodysplastic
syndrome (n=101)
Ctrl (n=11)
Urothelial carcinoma
(n=135)
Ctrl (n=52)
362.3 ± 84.1
331.9 ± 133.8
CSF (ng/ml)
Plasma
609.7 (127.6 – 1054.0)
197.1
434.86 ± 186.02
337.5 ± 71.4
57
Chapter 3: Characterization of Ang1 and Ang2 Protein Expression after
Acute Rat Spinal Cord Injury
3.1
Introduction
Upon injury, spinal cord pathology deteriorates via a secondary progressive cascade
of events that expands through initially undamaged spinal cord parenchyma (reviewed in
[364]). The vascular response following acute SCI is considered one of the major factors
implicated in the propagation of secondary injury. While it has been recognized for many
years that trauma to the spinal cord disrupts its local vasculature, researchers in the field are
now becoming increasingly aware that vascular dysfunction is a major contributor that
integrates the many components of secondary pathophysiology after SCI (reviewed in [365]).
The study described in Chapter 2 utilized CSF from acute human SCI patients as a
biological proxy for the biochemical events occurring within the spinal cord after SCI. As
Ang1 and Ang2 are secreted proteins, it was reasonable to hypothesize that their release from
pericytes and endothelial cells, respectively, would be detectable in CSF, which bathes the
CNS. Spinal cord tissue would contain the most relevant, most acute, and most concentrated
biological information regarding endogenous protein concentrations after acute SCI.
However, given that human spinal cord specimen can only be retrieved post-mortem, spinal
cord tissue samples from a commonly used contusion model of rat SCI was chosen for this
study. Furthermore, CSF quantity is limited in rats, and the effects of incubation at 37°C
(average human body temperature) in the intrathecal space are unknown.
The relationship between protein changes in CSF and spinal cord tissue remains to be
validated, and a direct comparison of Ang1 and Ang2 protein expression has never been
reported between the CSF compartment and the tissue of the spinal cord itself. The use of
58
CSF as a biological proxy for spinal cord tissue to study and evaluate the patterns of protein
expression, could further our knowledge of the feasibility of using CSF as a method of
sampling in future investigations of protein expression patterns in SCI patients, where spinal
cord tissue samples are not readily available.
Contusion injuries are considered the most clinically relevant model of SCI [366],
with traumatic, ischemic, and vascular components to the inflicted injury. In the current
study, the Infinite Horizon (IH) Spinal Cord Impactor was used to create a contusion injury at
thoracic levels T9 and T10 in rodents to model the human condition. The IH model is a
model of force-controlled impactor which has been gaining momentum on the SCI market in
recent years due to its ability to create consistent, reproducible contusion injuries [367-369].
After SCI is inflicted, spinal cord samples were collected to assess Ang1 and Ang2 protein
expression at acute and subacute time points post-injury.
This study seeks to address the question of what biochemical events are actually
occurring within the cord with regards to Ang1 and Ang2 protein expression. A comparison
between these findings and the ones presented in chapter 2 describing Ang1 and Ang2
protein expression in CSF after the acute human condition will further explore of the vascular
response after acute SCI.
3.2
3.2.1
Materials and Methods
Animals and Housing Conditions
All animal procedures were performed in accordance with the guidelines of the
Canadian Council for Animal Care and approved by the University of British Columbia
Animal Care Committee. Animals were group-housed prior to injury, and individually-
59
housed for 3 days post-injury then re-grouped. Housing facilities were set in a reversed 12hour day night cycle, and all animals had access to food and water ad libitum throughout the
duration of the study.
3.2.2
Surgical Procedures
63 adult male Sprague-Dawley rats (360 ± 50 g, Charles River Laboratories
International Inc., Wilmington, MA, USA) were randomized into 1 of 3 experimental groups:
SCI (n = 36), sham (n = 21), or naïve (n = 6) groups. Because the effect of laminectomy on
Ang1 and Ang2 protein expression were unknown when this study commenced, a sham
group (laminectomy only, no SCI) was included at each time point in this study, as well as a
naïve group (no laminectomy, no SCI).
SCI and sham animals were induced with 4% and anesthetized with 2% isoflurorane
gas in oxygen (1L/min). The surgical site was shaved and sterilized with repeating Betadine
and ethanol washes, and a local intramuscular injection of lidocaine with 2% epinephrine (33
mg/kg, Biomeda-MTC, Cambridge, ON) was administered. Subcutaneous injections of
buprenorphine (0.03 mg/kg, Temgesic ®, Rekitt Benkiser Healthcare Ltd., Berkshire, UK)
and 0.9% physiological saline (10 ml, lactated Ringer’s solution) were given prior to and
following surgery, and every 12 hours for 2 days post-surgery. A dorsal midline incision was
made to expose T8-T11 vertebrae and a T9-T10 midline bilateral laminectomy was
performed to expose the spinal cord and create a window for injury.
SCI animals (n = 36) were secured to the impactor device by clamping the T8 and
T11 spinal processes, and received a midline contusion using the IH Spinal Cord Impactor
(200 kdyn, Precision Systems and Instrumentation, LLC. KY, USA). Sham animals (n = 21)
60
were clamped to the impactor similarly, but no contact was made between impactor tip and
dura. After surgery, animals were kept in a 32°C humidified incubator until fully awake and
mobilizing. Naïve animals (n = 6) received neither SCI nor laminectomy.
Table 3.1
Sample population of experimental groups presented in the current study.
SCI
Sham
Naïve
3.2.3
2 hours
n=8
n=4
24 hours
n=7
n=4
48 hours
n=8
n=4
n=6
72 hours
n=7
n=4
120 hours
n=6
n=5
Tissue Collection
At five pre-determined end points (2, 24, 48, 72, and 120 hours post-injury), rats were
deeply anaesthetized with an intramuscular injection of ketamine hydrochloride (60 mg/kg,
Vetalar, Bioniche Animal Health Canada, Belleville, ON) and xylazine hydrochloride
(8mg/kg, Rompum, Bayer Inc. Etobicoke, ON) to the hind limb. Rats were then perfused
trans-cardially with 150 ml of cold phosphate-buffered saline (PBS), and a 0.5 cm section of
the T10-T11 spinal cord centred around the injury epicentre (or the corresponding spinal
level) was removed and snap-frozen in an ethanol-dry ice bath. Samples were stored at -80°C
until further processing.
3.2.4
Western Blot
The collected spinal cord tissue was homogenized in ice cold 0.01M PBS solution
containing a protease inhibitor cocktail (Cat # 11836153001, Roche Diagnostics GmbH,
Mannheim, Germany). Tissue samples were then centrifuged at 10000 rcf for 10 minutes at
4°C and the supernatant collected. Total protein concentration was determined for each
spinal cord supernatant sample using a standard bicinchoninic acid (BCA) protein titration
61
assay (Pierce Biotechnology, Rockford, IL, USA). A total of 20 µg of protein was heated at
95°C for 5 minutes before being loaded onto a 4% stacking and 12% resolving gel for
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and separation by
weight. Three gels were loaded with one randomly chosen sample each from each
experimental group (total of five SCI groups, five sham groups, and one naïve group). After
the temporal patterns of expression have been established, all samples from each
experimental group were loaded onto the same gel for comparison of inter-group variation.
Proteins were electrophorectically transferred to a polyvinylidene fluoride (PVDF)
membrane (Cat # ISEQ09120, Millipore, Billerica, MA, USA), blocked with a 5% bovine
serum albumin (BSA) and 1% Tween-20 solution for 1 hour at room temperature, and probed
with primary antibodies. Membranes were incubated overnight at 4°C with primary
antibodies against Ang1 (1:250, rabbit polyclonal, Cat # ab8451, Abcam, Cambridge, MA,
USA), Ang2 (1:500, rabbit polyclonal, Cat # ab65835, Abcam, Cambridge, MA, USA) or βactin (1:10000, mouse monoclonal, Cat # 691002, ICN Pharmaceuticals Ltd., Hercules, CA,
USA). Horseradish peroxidise (HRP)-conjugated secondary antibodies (1:10000, Goat antiRabbit HRP conjugate and Goat anti-Mouse HRP conjugate, BioRad, CA, USA; 1:10000,
Goat anti-Mouse HRP conjugate, Cedarlane, Burlington, ON) were used. Blots were
visualized using a standard chemiluminescent kit (Immun-Star ™ HRP Chemiluminescence
Kit, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s recommendations.
Detailed methodology can be found in Appendix B.
62
3.2.5
Quantification and Statistical Analysis
The intensity of protein bands were quantified using the Gel Analysis function in
Image J software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA.
http://imagej.nih.gov/ij). Expression of Ang1 and Ang2 were normalized to the expression of
β-actin of the same sample before being averaged amongst individuals of the same
experimental group. The number of individual animals in each group is presented in Table
3.1. Data is presented as mean ± SEM in arbitrary units (a.u.) after normalization to β-actin
expression.
Statistical analysis was performed using SPSS Statistics 18.0 software. Because data
did not show normal distribution, non-parametric tests were used. No significant differences
in Ang1 or Ang2 protein expressions were found between naïve or sham animals at any point
during the study, and thus those groups were combined into a single ‘uninjured’ group
henceforth for further analysis and discussion. Comparisons were performed at each time
point between SCI and uninjured (sham and naïve animals combined) animals using the
Mann-Whitney U-test. Statistical significance is reported at p < 0.05.
3.3
Results
The current study analyzes the temporal changes in Ang1 and Ang2 protein
expression in rat spinal cord after acute traumatic SCI. These rats were sacrificed at 2, 24, 48,
72, and 120 hours post-injury in order to examine the changes through time (Table 3.1).
Expression levels of Ang1 and Ang2 were determined by standard Western Blotting
techniques on homogenized spinal cord tissue supernatant.
63
Ang1 was detected as a single band with a molecular weight of ~40 kDa (Figure 3.1).
After SCI, there is a prominent decrease in Ang1 protein (Figure 3.2, Table 3.2). At 2 hours
after SCI, there is a 55% decrease in Ang1 protein levels when compared to uninjured
animals. Expression levels decreased from 2.10 ± 0.35 a.u. in uninjured animals to 0.77 ±
0.13 a.u. (p = 0.011, Mann-Whitney U-test) at 2 hours post-injury (Figure 3.2). Ang1
expression remained lower in SCI animals until 72 hours post-injury, when expression level
was 0.91 ± 0.17 a.u. (p = 0.081, Mann-Whitney U-test).
Figure 3.1
Representative image of Ang1 protein levels in rat spinal cord after acute SCI.
Image shows protein expression of Ang1 as detected by Western Blot. Lanes 1, 3, 5, 7, 9 show one SCI
animal each, at 2, 24, 48, 72, and 120 hours post-injury, respectively. Lanes 2, 4, 6, 8, 10 show one sham
animal each, at 2, 24, 48, 72, and 120 hours after laminectomy. Lane 11 is blank. Lane 12 shows a naïve
animal.
64
Figure 3.2
Quantification of Ang1 protein expression in rat spinal cord after acute SCI.
Expression levels as detected by Western Blots in uninjured rat spinal cords and at 2, 24, 48, 72, and 120
hours post-injury. Data is presented as group means ± SEM in a.u. as a ratio to β-actin expression.
Expression at 2 hours post-injury was 0.77 ± 0.13 a.u. whereas expression in uninjured animals was 2.10
± 0.35 a.u. * denotes p < 0.05 significant difference from uninjured group. # denotes p < 0.10 showing
trends in the data.
Ang2 was consistently detected as a single ~65 kDa band (as predicted by the
manufacturer) in the spinal cord of naïve and sham animals while a lower ~25 kDa band was
observed after SCI (Figure 3.3). Although the identity of this smaller peptide remains to be
confirmed, for simplicity, it will be referred to as ‘low molecular weight Ang2’ for the
remainder of this thesis, while 65 kDa Ang2 will be referred to as ‘high molecular weight
Ang2’. At 2 hours post-injury, there is a 47% decrease in high molecular weight Ang2 when
compared to uninjured animals (p = 0.052, Mann-Whitney U-test, Figure 3.4, Table 3.3).
There were no significant differences in the expression of high molecular weight Ang2
between SCI and uninjured animals at later time points. Nor were there any significant
65
changes in total (low and high molecular weight Ang2 peptides combined) Ang2 protein
levels (Figure 3.5, Table 3.4).
Figure 3.3
Representative image of Ang2 protein levels in rat spinal cord after acute SCI.
Image shows protein expression of Ang2 as detected by Western Blot. Lanes 1, 3, 5, 7, 9 show one SCI
animal each, at 2, 24, 48, 72, and 120 hours post-injury, respectively. Lanes 2, 4, 6, 8, 10 show one sham
animal each, at 2, 24, 48, 72, and 120 hours after laminectomy. Lane 11 is blank. Lane 12 shows a naïve
animal.
66
Figure 3.4 Quantification of 65 kDa (high molecular weight) Ang2 protein expression in rat spinal cord
after acute SCI.
Expression levels as detected by Western Blots in uninjured rat spinal cords and at 2, 24, 48, 72, and 120
hours post-injury. Data is presented as group means ± SEM in a.u. as a ratio to β-actin expression.
Expression of the high molecular weight Ang2 at 2 hours post-injury was 0.61 ± 0.13 a.u. whereas
expression in uninjured animals was 1.25 ± 0.19 a.u. # denotes p < 0.10 showing trends in the data.
67
Figure 3.5
Quantification of total Ang2 protein expression in rat spinal cord after acute SCI.
Expression levels as detected by Western Blots in uninjured rat spinal cords and at 2, 24, 48, 72, and 120
hours post-injury. Data is presented as group means ± SEM in a.u. as a ratio to β-actin expression.
There is the transient appearance of the low molecular weight Ang2 protein product
after SCI (Figure 3.6, Table 3.5). Maximal levels of low molecular weight Ang2 were
observed at 24 hours post-injury, at which time expression levels in SCI animals were greater
than 13-fold that of uninjured animals. Group mean at 24 hours post-injury was 1.78 ± 1.22
a.u. and 0.13 ± 0.04 a.u. (p = 0.001, Mann-Whitney U-test) in uninjured animals. Expression
levels of low molecular weight Ang2 in SCI animals then diminishes over the subsequent 96
hours until it is no longer significantly different from uninjured controls at 120 hours postinjury.
68
Figure 3.6 Quantification of 25 kDa (low molecular weight) Ang2 protein expression in rat spinal cord
after acute SCI.
Expression levels as detected by Western Blots in uninjured rat spinal cords and at 2, 24, 48, 72, and 120
hours post-injury. Data is presented as group means ± SEM in a.u. as a ratio to β-actin expression.
Expression of low molecular weight Ang2 at 24 hours post-injury was 1.78 ± 1.22 a.u. whereas expression
in uninjured animals was 0.13 ± 0.04 a.u. * denotes p < 0.05 significant difference from sham group.
The inter-individual variation was also verified within experimental groups. At 120
hours post-injury, a clean single band was observed for all samples (n = 6) at ~ 65 kDa
(Figure 3.7). While at 24 hours post-injury, all rats (n = 7) showed 2 distinct Ang2 bands near
65 kDa and 25 kDa (Figure 3.8). A much weaker band was also observed ~ 40 kDa.
69
Figure 3.7
Ang2 protein expression at 120 hours post-injury.
6 replicates showing the consistency of the (lack of) 25 kDa low molecular weight Ang2 at 120 hours postinjury. 20 µg of total protein was loaded into each lane. Antibody was diluted to 1:500. Membrane was
exposed for 1 minute.
Figure 3.8
Ang2 protein expression at 24 hours post-injury.
7 replicates showing the consistency of 25 kDa low molecular weight Ang2 at 24 hours post-injury. 20 µg
of total protein was loaded into each lane. Antibody was diluted to 1:500. Membrane was exposed for 1
minute.
70
Ang1 and Ang2 protein expression patterns were also established in different tissues
in the adult rat (Figures 3.9 and 3.10). Various peripheral tissues including heart, liver,
kidney, skeletal muscle (gastrocnemius muscle from hind limb), pancreas, skin, as well as
CNS tissue in the brain and chronic injured spinal cord (8 weeks post-injury, distal from
injury site) were probed. Pancreas and skin were chosen as negative controls, as these were
not expected to be sites of robust active angiogenesis.
Figure 3.9
Ang1 antibody tested on rat adult peripheral tissues and uninjured spinal cord.
Lane 1: Full-range rainbow ladder. Lane 2: Empty. Lane 3: Rat heart. Lane 4: Rat brain. Lane 5: Rat
liver. Lane 6: Rat kidney. Lane 7: Rat gastrocnemius muscle. Lane 8: Rat pancreas. Lane 9: Rat skin.
Lane 10: Rat uninjured thoracic spinal cord. 20 – 60 µg of total protein was loaded into each lane.
Antibody was tested in a 1:100 dilution in 5% BSA solution overnight at 4°C (note this is at a higher
concentration than the protocol used in chapter 3).
71
In non-CNS tissues, a strong band was detected at 55 kDa as predicted by the
manufacturer (Figure 3.9: lanes 3, 5, 6, 7), which appears to be the predominant isoform in
these tissues. Weaker bands were also observed at ~ 40 kDa and a band of variable weight
near ~31 kDa was sometimes observed (Figure 3.9: lanes 3, 5, 7). However, in the adult CNS
(brain and spinal cord), a much weaker detection of the expected 55 kDa band was observed
in comparison to a more dominant band at ~ 40 kDa (Figure 3.9: lanes 4 and 10).
The protein expression profile of Ang2 in various adult peripheral tissues was
similarly explored (Figure 3.10). Both low and high molecular weight Ang2 peptides were
detected consistently, although the intensity of each band in the various tissue types appear to
be unique (Figure 3.10).
Figure 3.10
Ang2 antibody tested on rat adult peripheral tissues and uninjured spinal cord.
Lane 1: Full-range rainbow ladder. Lane 2: Empty. Lane 3: Rat heart. Lane 4: Rat brain. Lane 5: Rat
liver. Lane 6: Rat kidney. Lane 7: Rat gastrocnemius muscle. Lane 8: Rat pancreas. Lane 9: Rat skin.
Lane 10: Rat uninjured thoracic spinal cord. 20 – 60 µg of total protein was loaded into each lane.
Antibody was tested in a 1:500 dilution in 5% BSA solution overnight at 4°C.
72
Because of the apparent varying weight of the low molecular weight Ang2 band
observed in these tissues, slight changes were made to the Western Blot protocol. Both Ang1
and Ang2 are glycoproteins (Figures 1.3 and 1.5), and it has been established that
deglycosylation of the protein samples may result in bands of differing weights. To address
this, two different rat spinal cord samples were subjected to a standard deglycosylation
process.
Interestingly, after deglycosylation, there is the appearance of another band ~31 kDa
distinct from 40 kDa Ang1. This downward shift could indicate the loss of glycan groups
from the native protein, resulting in a lighter peptide product (Figure 3.10: lanes 5 and 8).
Figure 3.11
and Ang2.
Ang1 antibody tested by different SDS-PAGE protocols and on recombinant human Ang1
Lane 1: Full-range rainbow ladder. Lane 2: Naïve. Lane 3-5: SCI 2 hpi. Lane 3: Denatured. Lane 4:
Without denaturation. Lane 5: Deglycosylated. Lane 6-8: SCI 24 hpi. Lane 6: Denatured. Lane 7:
Without denaturation. Lane 8: Deglycosylated. Lane 9: Recombinant human Ang1 protein (5 µg). Lane
10: Recombinant human Ang2 protein (5 µg). 20 µg of total protein loaded into lanes 2-8.
73
There have also been reports of dimeric (or higher order oligomers) yielding protein
bands of differing weights in SDS-PAGE as a result of the denaturation process [367-369].
To address this, the same two spinal cord samples were loaded for SDS-PAGE with and
without prior denaturation (Figures 3.11 and 3.12). No significant shifts in the protein
expression pattern were observed with or without denaturation (Figure 3.11: lanes 4 and 7
and Figure 3.12: lanes 3 and 6). To further confirm the characteristics of the antibodies used
in this study, recombinant human Ang1 and Ang2 proteins were also loaded for SDS-PAGE
(Figures 3.11 and 3.13). As predicted by the manufacturer, Ang1 antibody detected
recombinant Ang1 (and not Ang2) at ~70 kDa (Figure 3.11: lanes 9 and 10). Likewise with
Ang1, two spinal cord samples were subjected to deglycosylation, and probed for Ang2. No
remarkable changes were observed in the pattern of Ang2 protein expression in the spinal
cord after deglycosylation (Figure 3.12: lanes 4 and 7) or without denaturation (Figure 3.12:
lanes 3 and 6) of the protein sample prior to SDS-PAGE. Recombinant human Ang1 and
Ang2 proteins were loaded for SDS-PAGE (Figure 3.13). 5 µg of recombinant human Ang2
gave a strong signal at ~ 70 kDa (as predicted by the manufacturer), while 2.5 µg of Ang2
gave a weaker signal (Figure 3.13: lanes 3 and 4). Recombinant human Ang1 was not
detected by the Ang2 antibody (Figure 3.13: lanes 1 and 2).
74
Figure 3.12
and Ang2.
Ang2 antibody tested by different SDS-PAGE protocols and on recombinant human Ang1
Lane 1: Naïve. Lane 2-4: SCI 2 hpi. Lane 2: Denatured. Lane 3: Without denaturation. Lane 4:
Deglycosylated. Lane 5-7: SCI 24 hpi. Lane 5: Denatured. Lane 6: Without denaturation. Lane 7:
Deglycosylated.
Figure 3.13
Ang2 antibody tested on recombinant human Ang1 and Ang2.
Lane 1: Recombinant human Ang1 protein (2.5 µg). Lane 2: Recombinant human Ang1 protein (5 µg).
Lane 3: Recombinant human Ang protein (2.5 µg). Lane 4: Recombinant human Ang2 protein (5 µg).
75
Lastly, one membrane was simultaneously probed for Ang1, Ang2 and β-actin
(Figure 3.14). Ang1 and β-actin primary antibodies were incubated first. After detection, the
membrane was left to dry overnight. Ang2 primary antibody was incubated after 48 hours.
Distinct protein bands representing Ang1 (40 kDa), Ang2 (65 kDa and 25 kDa), and β-actin
(45 kDa) could be observed on this membrane (Figure 3.14).
Figure 3.14
The same membrane has be probed for Ang1, Ang2, and β-actin.
Membrane was first probed for Ang1 (rabbit polyclonal) and β-actin (mouse monoclonal) simultaneously.
Then stripped and re-probed for Ang2. 40 kDa band represents Ang1. 45 kDa band represents β-actin. 65
and 25 kDa bands represent Ang2.
Lane 1: Full-range rainbow ladder. Lane 2: empty. Lane3: SCI 2 hpi. Lane 4: Sham 2 hpi. Lane 5: SCI
24 hpi. Lane 6: Sham 24 hpi. Lane 7: SCI 48 hpi. Lane 8: Sham 48 hpi. Lane 9: SCI 72 hpi. Lane 10:
Sham 72 hpi. Lane 11: SCI 120 hpi. Lane 12: Sham 120 hpi. Lane 13: empty. Lane 14: Naïve.
20 µg of protein was loaded into each lane. Ang1 antibody was diluted 1:250. Ang2 antibody was diluted
1:500. β-actin antibody was diluted 1:10000.
76
Table 3.2
Ang1 protein expression in rat spinal cord after acute SCI.
Data is presented as group means (range) in a.u. as a ratio to β-actin expression.
Uninjured
2.10
(0.40 – 8.65)
Table 3.3
2 hours
0.77
(0.31 – 1.46)
24 hours
1.17
(0.22 – 2.17)
48 hours
1.83
(0.12 – 4.44)
72 hours
0.91
(0.07 – 1.46)
120 hours
3.05
(0.49 – 7.50)
48 hours
1.09
(0.05 – 2.54)
72 hours
1.16
(0.22 – 2.74)
120 hours
0.83
(0.13 – 2.26)
48 hours
1.60
(0.06 – 4.19)
72 hours
1.43
(0.40 – 3.17)
120 hours
0.98
(0.17 – 2.60)
72 hours
0.26
(0.07 – 0.77)
120 hours
0.16
(0.01 – 0.34)
65 kDa (high molecular weight) Ang2 protein expression in rat spinal cord after acute SCI.
Data is presented as group means (range) in a.u. as a ratio to β-actin expression.
Uninjured
1.25
(0.28 – 4.55)
Table 3.4
2 hours
0.61
(0.24 – 1.11)
24 hours
1.71
(0.21 – 6.78)
Total Ang2 protein expression in rat spinal cord after acute SCI.
Data is presented as group means (range) in a.u. as a ratio to β-actin expression.
Uninjured
1.33
(0.32 – 5.17)
Table 3.5
2 hours
1.21
(0.56 – 2.80)
24 hours
3.49
(0.93 – 15.84)
25 kDa (low molecular weight) Ang2 protein expression in rat spinal cord after acute SCI.
Data is presented as group means (range) in a.u. as a ratio to β-actin expression.
Uninjured
0.13
(0.01 – 0.69)
2 hours
0.60
(0.27 – 1.69)
24 hours
1.78
(0.17 – 9.05)
48 hours
0.51
(0.02 – 2.09)
77
3.4
Discussion
The study described in the current chapter investigates temporal changes in Ang1 and
Ang2 protein expression in the rat spinal cord after acute SCI. A sharp decrease in Ang1
protein levels was observed, when compared to uninjured animals. There is a transient
appearance of the low molecular weight Ang2 peptide product after acute SCI, but
surprisingly, no significant changes in total (low and high molecular weight) Ang2 band
intensities combined, Figure 3.5, Table 3.4) Ang2 expression.
A significant 55% decrease in Ang1 expression was observed 2 hours post-injury
(Figure 3.2). Similar to the current findings, there have also been reports of decreases in
Ang1 mRNA [343] and protein expression [337, 370] after acute SCI in rats. These decreases
in Ang1 were reported in mRNA from 6 hours to 7 days post-injury [343], and in protein
from 24 hours to 3 [370] and 8 [337] weeks post-injury. Together, these suggest that changes
in Ang1 after SCI are a dynamic process being actively regulated after SCI has been
inflicted. The sustained down-regulation of Ang1 after acute traumatic SCI suggests that
there may be prolonged impairments at endothelial cells junctions and in NVU integrity after
SCI, resulting in increased BSCB permeability [337, 370]. This is reflected in the seminal
works by Tator and colleagues studying vascular changes after acute SCI (reviewed in [180,
371-374]). Tator and Koyanagi reported vascular abnormalities in human spinal cord
specimen 9 months post-injury [157]. Popovich et al observed increased BSCB permeability,
analyzed via a radioactive vascular tracer administered intravenously, at 28 days post-injury
in rats [190]; while Risling et al reported a similar increase in BSCB permeability, analyzed
by HRP-conjugated vascular tracer administered intravenously, 7 months post-injury in
guinea pigs [189].
78
In contrast, a transient up-regulation of low molecular weight Ang2 was observed for
up to 3 days post-injury, peaking at 24 hours post-injury with a 13-fold increase in expression
compared to uninjured animals (Figure 3.6). Durham-Lee et al reported a decrease in Ang2
protein expression at 24 hours post-injury, and this study continued on to show significant
increases in Ang2 protein from 7 days to 5 weeks post-injury [370]. The presence of this low
molecular weight band at early time points post-injury (24 hours) and the absence of it in
uninjured, or at later time points post-injury (120 hours) is consistent amongst all animals in
those groups (Figures 3.7 and 3.8).
It can be hypothesized that the low molecular weight Ang2 band reported in the
current study could represent a secreted form (or what has already been secreted and is being
broken down and degraded in extracellular space) of Ang2. Ang2 is a secreted glycoprotein
which exerts its antagonistic effects on Ang1 by pre-occupation of their common receptor,
Tie2, without inducing downstream cellular effects. Because of this, Ang2 must be secreted
to be considered ‘active’. It is possible that the native form of Ang2 resides at 65 kDa, while
secreted form, which would be exposed to cleavage and degradation by extracellular
proteases, is detected at the low molecular weight of 25 kDa. Taking this into account,
observations in this study suggests that Ang2 is secreted at high levels following SCI, until
endothelial Ang2 storages are depleted, at which point Ang2 secretion is diminishes to a
significantly lower level limited by the rate of its production.
Ang2 is tightly regulated at the transcriptional level [237, 273, 279, 280, 283-285].
After transcription, Ang2 is stored in intra-endothelial cell Weibel-Palade bodies alongside
von Willebrand factor, a potent coagulant [274]. While inside Weibel-Palade bodies, Ang2
protein molecules have a very stable profile, lasting at least 16 hours after inhibition of
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mRNA production [274]. In contrast, secreted Ang2 exhibits peak expression at 20 minutes,
and is reduced to minimal detection levels merely 30 minutes after stimulation [274].
Secreted Ang2 has been reported to have a very short half-life [274]. The smaller molecular
size of the low molecular weight Ang2 peptide may represent a cleaved version of Ang2
which has been degraded by endogenous proteases after secretion, while they would have
been protected inside Weibel-Palade bodies prior to release.
In addition, there is an abundance of matrix metalloproteinase (MMP) cleavage
recognition sites on both Ang1 and Ang2 (Figures 1.3 and 1.5). After SCI, there is an acute
up-regulation of MMP-2 [375-377] and MMP-9 [378, 379]. Full length rat Ang1 contains 19
MMP-2 cleavage recognition sites and 3 MMP-9 cleavage recognition sites (Figure 1.3). Full
length rat Ang2 contains 34 MMP-2 cleavage recognition sites and 3MMP-9 cleavage
recognition sites (Figure 1.5). It is thus conceivable that the spinal cord samples collected in
the current study represent cleaved fragments of these proteins.
While low molecular weight Ang2 showed a strong up-regulation after SCI (Figure
3.6), the high molecular weight Ang2 band showed an early decrease protein expression at 2
hours post-injury (Figure 3.4). It is also important to note that the total amount of Ang2
(intensities of low and high molecular weight Ang2 peptides combined) did not show any
significant change throughout the study. Hence, the profound increase in low molecular
weight Ang2 reported could potentially be attributable to the substantial amount of Ang2 is
being released and broken down; while the activation of Ang2 production is initiated to
maintain the observed, relatively stable levels of total Ang2.Moreover, the timeframe in
which the low molecular weight Ang2 band stays elevated coincides with previous reports of
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increased BSCB permeability and inflammatory infiltration after SCI [144, 152, 156, 192,
274].
The instability of endothelial cell junctions and the BSCB has broader consequences,
particularly in the propagation of secondary injury after SCI. For instance, many cytotoxic
molecules such as calcium [146, 193], excitatory amino acids [194, 195], free radicals [196],
erythrocytes [144-146, 152, 157, 188, 197] are flushed into the injury penumbra, causing
further cell death. Inflammatory mediators [198, 380, 381], which are known to be acutely
up-regulated after SCI, also enter the injury penumbra, driving and reinforcing the
inflammatory cascade in a positive feedback loop through the release of inflammatory
cytokines to exacerbate damage in the injury penumbra.
This is the first report of the 40 kDa Ang1 protein band, observed in the current study.
The majority of research outside the CNS have reported the detection of Ang1 protein (in a
Western Blot) between around 55 – 75 kDa. Because of this discrepancy, confirmation of the
detected protein product was carried out in a series of adult rat CNS and peripheral tissues
(Figure 3.9). At least two products, one approximately 55 kDa and the other approximately
40 kDa were consistently observed. However, the relative intensity of these two products
appeared to shift in the various tissues. In peripheral tissues, the 55 kDa product (as predicted
by the manufacturer) was most prominent; while in CNS tissue (brain and spinal cord), the
intensity of the 55 kDa product was inferior to the signal intensity of the 40 kDa product.
Because of the unique patterns of expression seen in different tissues, it was
hypothesized that there may be differences in isoform expression or post-translational
modifications of Ang1 that occurs in the various types of tissue. Such differential patterns of
expression of different protein isoforms have been reported for VEGF, a protein closely
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related to the Angiopoietin family. Different isoforms of VEGF have been reported to be
expressed in different tissues, which have differential functions in the wide spectrum of
processes that constitute the angiogenic process [382-386]. Differential isoform expression of
VEGF has been reported during different phases of developmental and adult angiogenesis
[385], CNS development [387], following TBI [388], and after hypoxic episodes [389].
Another possible explanation for the discrepancies in the protein weight of Ang1
reported in literature and the one detected in the current study could be due to the different
mRNA splices that exist [390]. These different splices of the Ang1 transcripts translate into
Ang1 proteins of different protein sizes [390]. Huang et al identified a 40 kDa Ang1 peptide
in CHRF cells (megakaryocyte cell line), which was identified as the 0.9 kb splice variant of
Ang1, while the 1.3 kb and 1.5 kb variants resulted in 55 and 65 kDa bands in a similar
Western Blot protocol [390]. Both the 40 and 65 kDa were shown to bind to and interact with
Tie2 receptor in vitro, while the 55 kDa isoform did not [390].
Different mRNA splices of Ang2 has also been reported [391], although this 25 kDa
isoform appears to be novel. Likewise with Ang1, its expression was explored in different
peripheral and CNS tissues (Figure 3.10). Both high and low molecular weight Ang2
products were detected consistently in all of the probed tissue types, though the relative
signal intensity of each in the different types of tissue was different (Figure 3.10).
Durham-Lee et al have recently reported on the up-regulation of Ang2 protein
expression for up to 70 days post-injury in another rat contusion SCI model [370]. Although
the antibody used in this study was the same as the one used in the current study, this study
reported a single band at ~70 kDa, and noted the presence of dimers in their Western Blot
analysis, which was not detected in the current study. These variations could be attributed to
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differences in protocol between that study and the current one. In their study, samples were
not denatured prior to loading for SDS-PAGE, whereas the current samples were heated at
95°C for 5 minutes for denaturation. Heating protein samples prior to SDS-PAGE facilitates
the unfolding of the protein tertiary structure. Loading a folded protein for SDS-PAGE could
affect their migration down the gel, as folded proteins often carry charges on them. This
difference in protocol was examined with the Western Blot protocol used in the current study
(Figures 3.11 and 3.12). There appears to be no differences in the migration of neither Ang1
nor Ang2 in SDS-PAGE due to protein denaturation (Figures 3.11 and 3.12).
Additionally, Ang1 contains 5 glycosylation sites and Ang2 contains 6 (Figures 1.3
and 1.5). There may be differential glycosylation patterns in different tissues, leading to
slightly different apparent protein weights. These potential differences were explored with
the addition of PNGase to remove glycan groups from the collected spinal cord samples.
PNGase F (Cat # P0704S, New England Biolabs Inc., Pickering ON) was incubated with 20
µg of spinal cord samples from 2 and 24 hours post-injury to remove glycan groups.
Deglycosylation of injured spinal cord samples resulted in a downward shift in the apparent
molecular weight of Ang1 (Figure 3.11). This is consistent with the loss of glycan groups
from a peptide, resulting in a lighter/smaller protein product. However, no such shift was
observed when probed for Ang2 after deglycosylation (Figure 3.12).
Recently, concerns have been raised about the specificity of the polyclonal Ang2
antibody used in the current study. There have been reports on the cross-reactivity of this
antibody in the tissue of Ang2-knockout mice. To address the issue of antibody specificity,
the antigen that each antibody (both Ang1 and Ang2) was raised on were aligned on basic
local alignment search tool (BLAST) against the rat genome and confirmed to be specific. In
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addition, purified full-length recombinant human Ang1 and Ang2 proteins were assessed by
SDS-PAGE and probed with both Ang1 and Ang2 primary antibodies. Recombinant human
Ang1 (5 µg) was detected by the Ang1 antibody at ~70 kDa, as predicted by the
manufacturer (Figure 3.13). This protein was not detected by the Ang2 antibody (Figure
3.13). Recombinant human Ang2 (2.5 and 5 µg) was detected only by the Ang2 antibody,
also at ~70 kDa (Figure 3.13). One membrane was also re-probed for both Ang1 and Ang2
antibodies, and no overlaps were found in the band heights of Ang1, Ang2, and β-actin
(Figure 3.14). Together, these suggest that the protein bands detected in the current study
may differ from the native (human) protein due to physiological modification and/or
degradation, or as a result of the sample processing process. Further differences could also
exist in mice tissue which may influence the cross-reactivity between Ang1 and Ang2 with
this antibody. Of note, the antigen that this antibody was raised on is a short recombinant
peptide that is predicted to interact with human and rat (but not mouse) Ang2.
The use of spinal cord tissue would give a more accurate profile of the temporal
changes in these proteins as they are expressed and secreted by spinal cord vasculature. But
because the biochemical techniques used in this previous study (ELISA) was notably
different than the current one (Western Blot), this makes comparison with the data presented
in chapters 2 and 3 difficult. The low molecular weight band detected in the current study
would not have been detected in the human CSF due to the inability of the ELISA technique
to distinguish protein isotypes or weights. Furthermore, because there is little cellular content
in CSF, the molecules of Ang2, which were stored in WP-bodies inside endothelial cells,
may not have been detected at all.
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In order to validate the hypothesis that low molecular weight Ang2 may be a secreted,
active form of the protein, mRNA expression of Ang2 could be characterized in future
experiments. Furthermore, because of the reported roles that Ang1 and Ang2 have on the
nervous system and their expression in non-vascular cells, the ability to locate Ang1 and
Ang2 protein expression by means of immunohistochemistry could provide further details
regarding the activity of these proteins after SCI. As this study reports on the protein
expression profiles of Ang1 and Ang2 with novel patterns of expression after Western
Blotting, further validation of these novel protein bands can be confirmed with sequencing by
mass spectrometry. Functional assays utilizing immunoprecipitation with Tie2 receptors
could determine whether these protein peptides will bind to endothelial Tie2 to elicit
downstream effects.
As the understanding of the pathophysiology of SCI progresses, the characterization
of vascular and angiogenic changes, as well as the role that they play to orchestrate
secondary injury after SCI will add to, and help integrate the many aspects of secondary
injury after SCI.
3.5
Conclusions
The current study illustrates the temporal progression of Ang1 and Ang2 in the spinal
cord of rats after acute SCI. Some similarities were observed between the patterns of Ang1
and Ang2 protein expression in the current study to those presented in chapter 2 in human
CSF, for example, the sustained increased in Ang2 expression in human CSF and rat spinal
cord tissue. Some differences were also noted, namely a down-regulation of Ang1
immediately after SCI in rat spinal cord while an early transient increase was observed in
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human CSF. Evidently, a direct comparison between the current study utilizing spinal cord
tissue and the previous one utilizing CSF should not, and cannot simply be made simply. If
the relationship between the CSF representing a biological proxy for biochemical events
occurring within the cord could be clarified, CSF could be a great tool for future clinical
studies, where obtaining cord tissue samples can only be done post-mortem. Comparisons
drawn between data presented in this and the previous chapter should be interpreted
cautiously with the consideration that there are profound (and understandable) differences in
many aspects of the biology, as well as the techniques utilized in these two studies presented
in chapters 2 and 3.
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Chapter 4: Integrated Discussion and Research Conclusions
This concluding chapter is an integrated discussion of the overall objectives presented
in this thesis. A summary of the critical findings of this thesis work, and its contribution to
the understanding of the vascular pathology of SCI will be presented. This will be followed
by a discussion of the future directions in which this project, and vascular research in
general, may and could take to further the understanding of the mechanisms of secondary
damage after SCI or other CNS pathologies. The subsequent section will then describe a
selection of the strengths and limitations of this thesis work, and address some long-standing
questions in the field regarding the translation of therapies from bench to bedside. This
chapter will close with concluding remarks and a re-examination of the overall objectives of
this thesis.
4.1
Summary of Findings
This thesis presents novel findings in the endogenous protein expression of Ang1,
Ang2, and Angiogenin (chapter 2 only) in human CSF and serum, and rat spinal cord
homogenate after acute SCI. Multiple time points were analyzed in both studies, thus
allowing for an examination of the temporal progression of change in this series of
angiogenic proteins.
In the first part of the thesis (study #1 presented in chapter 2), protein expression of
Ang1, Ang2, and Angiogenin were measured in CSF of acute SCI patients from 24 to 120
hours post-injury by commercially available sandwich ELISA kits. In the second part of the
thesis (study #2 presented in chapter 3), the protein expression of Ang1 and Ang2 were
measured in spinal cord tissue after acute SCI in rats, from 2 to 120 hours post-injury, by a
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standard Western Blot technique. Together, these works add to the growing body of literature
investigating vascular dysfunction and changes in BSCB permeability after acute SCI.
After acute human SCI, there is an initial spike in Ang1 levels in the CSF, which was
hypothesized to be the spillage of Ang1 molecules from spinal cord microvasculature due to
the mechanical impact. A subsequent increase in Ang2 at 36 hours post-injury coincides with
Ang1 decreasing back down to control levels, while Ang2 stays elevated. This pattern is
reflected in rat spinal cord tissue at 2 hours after acute SCI. In rats, there is an immediate
decrease of Ang1 protein expression in the spinal cord after SCI. This is compounded by a
subsequent 13-fold increase of low molecular weight Ang2.
The similar pattern of change can be seen between these two studies (Figures 4.1 and
4.2). Most prominently is the sustained up-regulation of Ang2 that was observed in both
human CSF and rat spinal cord. Low molecular wegiht Ang2 in the rat spinal cord was
elevated through the first 3 days post-injury, while Ang2 in human CSF saw a delayed
increase from 48 hours until at least 120 hours post-injury (Figure 4.2). Although the time
frames of these changes are not identical, both time frames coincide with the development of
inflammation and edema after SCI [198]. Ang1 protein expression goes through an early
decrease in rat spinal cord, and in human CSF, expression levels are similar to non-SCI
controls except for the first time point. While these are not the similar expression patterns, it
can be speculated that there is minimal Ang1 agonistic signalling to elicit downstream effects
on angiogenesis, cell-cell integrity, and cell survival (reviewed in [365, 392]). The shifts in
the time frame of the temporal patterns of Ang1 and Ang2 expression may be attributed to
the different medium and/or different species used in the respective studies. Although CSF is
a promising media to study biochemical changes in the CNS after SCI, the exact relationship
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between specific proteins in these two medium has not yet been established. Until this
relationship can be validated, CSF is merely a sampling of the proteins present in the
extracellular space within the thecal sac at the lumbar region (where CSF drains were
installed), and may not be fully representative (on both a concentration and time scale) to the
acute biochemical events occurring at the injury epicentre. The metabolic differences
between the two species, which may affect the temporal progression of the pathophysiology
and recovery processes, should also not be undermined.
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Figure 4.1
Relative expression of Ang1 in human CSF and rat spinal cord after acute SCI.
Aside from the early (but opposite) fluctuations, Ang1 protein expression appears to stay reasonably
stable (when compared to non-SCI or uninjured control levels) throughout the first 120 hours postinjury.
Figure 4.2 Relative expression of Ang2 in human CSF and rat spinal cord (low molecular weight) after
acute SCI.
Ang2 expression in both human CSF and rat spinal cord saw a sustained increase in the first 120 hours
post-injury.
4.2
The Role of Angiogenic Proteins in Vascular Disruption after Spinal Cord Injury
Vascular dysfunction after SCI has a central role to the propagation of secondary
damage after SCI. One of the major results of the vascular damage after SCI is the extensive
breakdown of the BSCB [152, 164, 188-192]. After SCI, the lack of such a barrier protecting
the injury penumbra results in the expansion of damage into previously uninjured tissue in
the injury penumbra. Increased BSCB permeability has been reported in the first 3 days after
SCI [144, 152, 156, 192]. This early peak in vascular leakage has been reported to coincide
with the acute inflammatory response [198], implicating the role of vascular permeability in
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the propagation of the inflammatory response after SCI. Between 3 and 7 days post-injury
[147, 156, 164, 188, 190, 199], initiation of angiogenesis and revascularization is reported at
the injury epicentre [148, 151, 154-156], and is followed by the restoration of the BSCB
[156, 188, 190]. Destabilization of existing vascular networks, could further exacerbate
secondary damage by interrupting perfusion to the injury penumbra. However, like many
physiological reactions to various injuries, increased vascular permeability after SCI is also a
reparative process. Increased vascular permeability increases endothelial plasticity and is a
pre-requisite for vascular remodelling to occur. This phase of angiogenesis is evident as a
breach of tight junctions, displacement of astrocytic foot processes, and separation of the
basement membrane [152, 188, 190].
Ang1 and Ang2 are important players in the regulation of the balance between
vascular quiescence and stability. High levels of Ang1 after SCI could serve to limit the
progression of inflammation, which is well-known to exacerbate functional deficits [190,
198] and also restrict the passage of cellular toxic molecules into the injury penumbra after
SCI. This was not observed in human CSF (with the exception of the 24-hour time point) or
in the rat spinal cord after SCI. In contrast to Ang1, high levels of Ang2, the natural
antagonist of Ang1, would result in the opposite effects; increasing BSCB instability, but
allowing the initiation of angiogenesis.
Ang1 is also a potent pro-survival factor for endothelial cells, and could help promote
their survival after SCI, thereby preserving perfusion in the injury penumbra to ameliorate
the cascade of cell death caused by metabolic stress at sites distal from the injury epicentre.
Elucidation of the specific mechanisms of Ang1and Ang2 signalling in the spinal cord could
further our understanding of the effects of Ang1 and Ang2 on BSCB permeability in
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quiescence and after SCI. Furthermore, the role of cells such as pericytes, and its relationship
to the interactions between the vascular and nervous systems under such circumstance may
be crucial to how repair and regeneration can be achieved after SCI. Ang1 signalling has also
been reported to elicit considerable pro-survival effects on neurons [326] and NPC [329].
The combination of low Ang1 and high Ang2 levels suggests that substantial
antagonistic effects may be exerted on downstream functions of Tie2 signalling to relax
endothelial cell junctions and increase BSCB permeability. As Ang1 has been reported to
have significant roles in the survival of both vascular and nervous cells [233, 238, 239, 329],
the changes in Ang1 and Ang2 expression after SCI could also be implicated in the
progressive cell death that is observed after acute SCI [145, 149, 153, 161, 162, 185-187,
340].
In context of the current research, the past 2 years have definitely seen increasing
interest in not only the use of vascular growth factor, including Ang1, as a treatment for SCI
[336, 337]. It is apparent that scientists are now more aware that modulating vascular
processes after SCI could result in significant long-term behavioural/neurological benefits.
Indeed, the application of Ang1 or other vascular growth factors as a therapy aimed to
improve neurologic outcome after SCI has also been investigated. Ang1, VEGF, and C16 (a
αvβ3 integrin peptide which binds to laminin) have all shown positive effects when
administered after experimental SCI [336, 337]. The combination of Ang1 and C16 resulted
in sustained functional improvements (Basso Mouse Scale, from ‘extensive ankle movement’
to ‘weight-bearing, consistent plantar stepping with some coordination’) in mice, and rescued
both vasculature (LEA, Lycopersicon esculentum lectin [393]) and white matter (Eriochrome
cyanine) at 42 days post-injury [336]. The combination of Ang1 and VEGF in rats improved
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vascular stability (MRI) and hind limb function (Basso-Beattie-Bresnahan locomotor rating
scale, from occasional to frequent coordination of steps) at 56 days post-injury [337]. The
lack of Ang1 protein expression reported in the current study, in conjunction with the
reported roles that Ang1 has on survival and BSCB integrity, supports these hypotheses that
an exogenous source of Ang1 could be beneficial after acute SCI.
4.3
Implications for the Future
The notion of integration between vascular and nervous systems in various
neurological pathologies has taken the spotlight in CNS disorder research. This is highlighted
in a recent issue of Nature Neuroscience, with a focus section dedicated to the investigation
and discussion around neurovascular interactions in both health and pathological conditions
[394]. Vascular dysfunction and BBB/BSCB abnormalities have recently been brought to
attention in a variety of CNS pathologies including ALS [28, 30], multiple sclerosis [33, 395398], Alzheimer’s disease [31, 32, 399-403], Parkinson’s disease [400, 401, 404, 405], and
stroke [406]. This increased attention to the neurovascular niche has brought on a series of
articles aimed at deciphering the role and function of a crucial, but previously undercharacterized component of the NVU: the pericyte. While previously considered stagnant
players in the NVU, pericytes have since been implicated in many crucial roles in the
development and maintenance of both vascular and nervous systems. Angiogenesis [102,
103, 108, 111], regulation of the BBB [33, 91, 98-100, 102, 108, 252], maintenance of
vascular stability and homeostasis [91, 92, 101], and the dynamic adjustment of blood flow
[33, 37, 114] have all been attributed to pericyte function.
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Advancement in vascular tracing [407-411] and imaging technology [412-421] has
led to better resolution of the cellular and vascular abnormalities after SCI, and a renewed
interest in the characterization of BSCB permeability and vascular dysfunction after SCI
[151, 164, 422, 423]. A more thorough understanding of the pathogenesis of secondary
damage may not only help to guide the discovery of intervention strategies to prevent or
attenuate these mechanisms, but may also provide new targets for which these strategies can
be aimed at alleviating paralysis caused by SCI.
The ability to use a substitute for spinal cord tissue such as CSF, for the interpretation
of changes occurring within the cord could provide a novel method for studying acute human
CNS injuries where extraction of CNS tissue for investigation is not ethically possible.
Furthermore, the comparison between the acute physiological processes that occurs after
human and rat SCI has allowed for the further comparison and validation of commonly used
rodent models of SCI (such as the thoracic contusion model used in this thesis work), to the
acute clinical condition.
Furthermore, neither technique employed in these two studies allowed for the
examination of the location in which the proteins are found. Another technique such as
immunohistochemistry may elude the location in which these proteins of interest are found
within the spinal cord parenchyma. Identifying the location of Ang1 or Ang2
expression/secretion may give further indication of the angiogenic status or the state of
vascular stability after SCI. There have also not been any reports on the correlation between
Angiopoietin levels and actual changes in BBB or BSCB permeability.
Finally, although extensive care has been given to ensure the validity and rigor of the
works presented, it is understandable that there are several technical issues while have made
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interpretation of the data presented difficult. One of the remaining questions which have not
yet been answered conclusively, revolves around the identity of the protein products detected
in chapter 3. The identity of the protein products should be confirmed with mass
spectrometry protein sequencing, which will enable the comparison between the detected
protein product and the known sequence of Ang1 or Ang2 (or if it turns out that they are
neither of these proteins, then what exactly are they). Functionally, the ability of these
protein products to interact or bind to the Tie2 receptor can be examined through an immuneprecipitated Western Blot. With VEGF, for example, different isoforms of VEGF have
different functions in vivo [382-386]. It would be interesting to be able to determine if
different isoforms (if indeed there are different isoforms) of Ang1 and Ang2 will behave in a
similar fashion.
4.4
The Translation Highway
The two studies presented in this thesis represents an example of an investigation into
a specific set of proteins undertaken in two different species, using two different biological
specimen, and two different molecular techniques; and exemplifies the need to interpret these
kinds of data with caution when trying to draw comparisons to the acute human condition for
translational purposes. Although the vast majority of the scientific understanding of the
pathophysiology of secondary damage after SCI is derived from such animal studies,
historically, therapies that have been shown to be effective in rodent models of SCI have not
been successful at demonstrating convincing neurologic benefits when translated to human
clinical trials. While there are many potential reasons for this, one is that critical biological
differences may exist between the pathophysiology of such commonly used animal models
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and that of the acute human condition and alludes to the fact that not only is our
understanding of the massively complex pathophysiology of SCI incomplete. The current
basis of translation rests on the assumption that the injury and recovery mechanisms between
commonly used rodent (or other animal) models and the acute human condition are
comparable in nature. Unfortunately, very often, promising results shown in animal models
not worked in human studies. This is particularly evident in the field of stroke research,
where a recent report has exhaustively reviewed a total of 1026 neuroprotective strategies,
many of which have been translated into the clinic, but none of which were decidedly
effective [424]. This speaks to the disquieting possibility that important biologic differences
do exist between these two conditions.
The current work shows that the temporal progression of Ang1 and Ang2 protein
expression appears to have at least some similarities between the two species, at least during
the first 5 days post-SCI. However, this is not the case for many inflammatory cytokines
[425, 426]. Although animal models are, and will continue to be a critical aspect of
biomedical research, this highlights the fact that our understanding of the pathophysiology of
SCI in neither human nor rats is not yet entirely complete. The reality that biologic
differences exist between the pathophysiology of animal and human SCI will be one hurdle
for researchers to overcome. A model using a larger animal with greater anatomic and
biologic similarities to humans could help to overcome this hurdle in validating preclinical
results and facilitate a successful translation from bench to bedside. Indeed, many (70%) in
the SCI field agrees that efficacy in a large animal model should be required before
traversing the translational leap [366].
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The other major challenge in SCI translation is the difficulty in objectively defining
‘clinically relevant’ outcome in preclinical studies. To interpret particular outcome measures
in animal models as meaningful neurologic improvement in a human would be unfair.
Identifying ‘clinically relevant’ outcomes is crucial in determining the predictive value of a
potential treatment. Besides the differences in the mode of locomotion between human and
most other model species, the dexterity, gross and fine motor function, and dependence on
our upper extremities, and the neuroanatomical characteristics, SCI for human encompasses a
wide array of behavioural, functional, and physiological effects which are poorly (if at all)
characterized in animal models, but hold remarkable place in the quality of life of SCI
patients. According to a survey carried out by Anderson et al, many of the factors which
were seen as important to SCI patients [427] are not those which are conventionally
measured in animal models. These include cardiovascular health and autonomic dysreflexia,
bladder and bowel function, and sex-related issues [427, 428]. Likewise, much of the clinical
research in SCI are heavily based upon quality of life scores or activities of daily life
instruments, which of course would not be applicable in animal models [429]. Any changes
in these secondary (to functional benefits defined by the AIS) could greatly improve the wellbeing of SCI patients, yet are not routinely studied in pre-clinical investigations. Identifying
appropriate primary and secondary outcomes in clinical trials is crucial. For example, in
addition to changes in AIS scores, perhaps the conductivity of spared or ‘repaired’ axons
could also be measured [430]; in cases where neurological improvements after treatment is
not enough to mount to a change in AIS grading.
We understand now that it is unlikely that any specific neuroprotective drug will be a
‘magic bullet’, and that ‘significant improvements’ will likely be measured in terms of
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relatively small gains in motor/sensory function in a modest number of patients.
Additionally, we understand much better now the vexing extent of variability in spontaneous
neurologic recovery, particularly in patients assessed very early after injury. These two issues
– the anticipation of a relatively small effect size and the recognition of high variability in
spontaneous neurologic recovery – mandate that fairly large numbers of patients be enrolled
in clinical trials.
The challenge of discovery science in pre-clinical research, the challenge to collect
sufficient evidence of efficacy, and finally, the substantiation of such experimental therapies
into clinical trials; is an exceedingly challenging, extremely expensive, time-consuming, and
laborious series of events. And even if the primary outcome is not positive, all is not lost. The
reverse-translation of data gathered from clinical studies back to the controlled environment
of the laboratory could be crucial to extract the difference that exists between animal models
and the clinical setting, and accelerate the tedious process of clinical translation of
pharmaceutical interventions for the treatment of SCI. Such guidance is invaluable in
planning subsequent clinical trials. And in addition to the drugs that have entered into clinical
trials, a handful of drugs are being actively explored because of their long-standing track
record for safety in human patients. The fact that clinically used compounds may also have
potential neuroprotective properties introduces the possibility for such therapies to enter
clinical trials with fewer safety concerns.
The appreciation for the complexity of SCI pathophysiology that has emerged over
the past few decades of valiant research effort has led to the belief that a multifaceted,
interdisciplinary combination of strategies will be necessary to treat paralysis arising from
SCI. Such strategies will take on multiple targets of treatment including (but not limited to)
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minimizing secondary damage, promoting plasticity of residual neural tissue and stimulating
axonal regeneration to reinnervate distal targets. Nonetheless, any neuroprotective strategy to
alleviate secondary injury mechanisms or any small gains of axonal conduction from
regeneration strategies may yield functionally relevant neurologic recovery that will improve
the lives of SCI patients tremendously.
4.5
Conclusions
At the conclusion of this thesis work, I want to highlight once again the critical
importance of integration of vascular pathology in the study of SCI. The pathophysiology of
SCI is not limited to a single, or two, or even three localized events occurring at the site of
injury, but rather, a systemic condition implicating the body as a whole. Interactions between
the vascular and nervous systems are especially of utmost importance.
This thesis work presents novel findings in the characterization of the endogenous
protein expression of Ang1, Ang2, and Angiogenin in CSF after acute human SCI. In
addition, Ang1 and Ang2 protein expression were also examined in the spinal cord of a rat
model of acute SCI. The notion that the vascular and nervous systems should be considered
not separate entities but a single integrated ‘unit’, is central to the interpretation of the
findings and conclusions drawn from the studies presented in this thesis work.
Looking towards the future, the validation as well as further investigation into the
techniques employed in this thesis work will help to advance the exploration of Ang1, Ang2,
and Angiogenin in CSF and tissue samples for SCI and other pathologies alike. The
‘translational highway’ from bench to beside, requires enormous time, money, and labour
costs. Many promising therapies fall off this highway at every stop. To help traverse this
99
translational curve, it is important to objectively critique pre-clinical studies presented in
animal models. Furthermore, to be able to choose the correct animal and injury model that
will be correctly represent the pathophysiology of SCI. To this note, the current thesis work
presents two similar studies conducted in two very different settings, highlighting the
difficulties in the extrapolation of pre-clinical data to the acute human condition.
As presented in the previous sections, the lack of a clear understanding of SCI
pathophysiology in both animal models and in human illustrates the vexing need to enhance
the bi-directional communication between the bench and bedside.
100
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124
Appendices
Appendix A Supplementary Information from Chapter 2.
A.1
Demographics and Medical Records of Human Subjects in Chapter 2.
Table S.1
Subject
SCI subjects enrolled in human clinical trial.
Age/Sex
Mechanism of
Injury
SCI 1
19/F
Fall
SCI 2
22/M
Transport
SCI 3
25/M
Sports
SCI 4
51/M
Fall
SCI 5
55/M
Sports
SCI 6
42/M
MVA
SCI 7
60/M
Fall
SCI 8
20/M
SCI 9
38/M
SCI 10
25/M
Transport
Struck on back of
head
Sports
SCI 11
64/F
Sport
SCI 12
66/M
Fall
AIS
Diagnosis
Motor Score
Last Normal
Sensory Level
Ad
1yr
Ad
1yr
Ad
1yr
A
A
50
50
T9
T7
A
A
60
66
L2
L2
A
C
43
58
C7
C7
A
A
50
50
T11
T11
A
--
27
--
C6
--
B
B
19
27
C5
C6
B
C
20
49
T9
C4
B
D
6
80
C5
C4
C5
B
C
30
86
C6
T6
C5 3 column # dislocation - closed
C5-6 hyperextension, avulsion
flakes, traumatic disc
C4-5 hyperextension, avulsion
flakes, traumatic disc
B
D
17
15
C5
C6
C
D
41
98
C5
C2
C
C
5
71
C2
C5
T10 flexion/distraction injury mixed - closed
L1 burst # - closed
C7 3 column burst # without
dislocation - closed
T10 translational injury - bony
(#/dl) - closed
C6-7 bilateral facet dislocation
C6-7 3 column burst # without
dislocation - closed
C4-5 3 column # dislocation closed
C6-7 bilateral facet dislocation
125
Subject
Age/Sex
Mechanism of
Injury
SCI 13
46/M
Transport
SCI 14
39/M
Sports
SCI 15
54/M
Transport
Table S.2
AIS
Diagnosis
C4-5-6 hyperextension, avulsion
flakes, traumatic disc
C3-4 hyperextension, avulsion
flakes, traumatic disc
C5-6 hyperextension, avulsion
flakes, traumatic disc
Motor Score
Last Normal
Sensory Level
Ad
1yr
Ad
1yr
Ad
1yr
C
D
33
83
C3
C4
C
D
36
65
C4
C5
C
B
13
--
C4
C4
Non-SCI control subjects enrolled in human clinical trial.
Subject
Age/Sex
CTRL 1
CTRL 2
CTRL 3
CTRL 4
CTRL 5
CTRL 6
49/F
85/F
52/F
68/F
45/M
61/F
Mechanism of
Injury
-------
CTRL 7
62/M
--
CTRL 8
59/M
--
Diagnosis
AIS
L4-5 recurrent disc herniation
L2-3, L3-4, L4-5, L5-S1 stenosis
Right S1 radiclopathy
Degenerative L3-4 spondylolisthesis
L5-S1 disc herniation
L5-6 disc herniation
L5-S1 degenerative disc disease with neural
foraminal stenosis
L3-4 spinal stenosis; L4-5 degenerative
spondylolisthesis
---------
126
Appendix B Supplementary Methodology from Chapter 3.
B.1
Determination of Protein Concentration
Protein concentration in spinal cord tissue homogenate was titrated against 2.5 µg/µl to 0.062
µg/µl standards of BSA solutions. 5ul of each homogenate sample were plated in duplicate in
200ul Pierce Protein Assay (1: 50 reagent A-to-reagent B) solution (Thermo Scientific, Cat #
23228, Rockford, IL, USA). The plate is then incubated at 37°C for 30 minutes and the
optometric density read at 570 nm.
B.2
SDS-PAGE
Tissue homogenate samples were diluted to 1mg/ml, and 20 µg of total protein (as
determined by BCA assay) was mixed with equal volume of 2 X Laemmli solutions and
heated to 95°C for 5 minutes to denature.
Table S.3
62.5 mM
25%
2%
0.01%
350mM
Laemmli buffer preparation.
2X Laemmli buffer
Tris-HCl (pH6.8)
glycerol
SDS
Bromophenol blue
DTT
Samples were loaded onto a 12% resolving with 4% stacking mini-gel for SDS-PAGE.
Table S.4
Stacking and resolving gels for SDS-PAGE preparation.
4% stacking gel 12% resolving gel
(ml)
(ml)
40% Acrylamide
1
3
2%
Bisacrylamide
0.53
1.6
1.0 M Tris pH 8.8
-3.75
1.0 M Tris pH 6.8
1.25
-127
20%
10%
SDS
ddH2O
Temed
APS
4% stacking gel 12% resolving gel
(ml)
(ml)
0.05
0.05
7.06
1.5
0.01
0.005
0.1
0.1
Gels were ran in 1X running buffer at 90V for 15 minutes then increased to 150V for
approximately 90 minutes (pending the progression of the dye band).
Table S.5
Running buffer preparation.
1X Running buffer
ddH2O
1L
Tris-Base
3.03 g
Glycine
14.42 g
SDS
1g
Electrophoretic transfer was performed at 100V for 2 hours at room temperature (with
apparatus surrounded by ice). Proteins were transferred to a PVDF membrane set between
layers of fiber pads, Whatman filter paper, submerged in 1X Transfer buffer.
Table S.6
Transfer buffer preparation.
ddH2O
Tris-Base
Glycine
MeOH
1X Transfer buffer
800 ml
2.32 g
11.6 g
200 ml
128
After transfer, membranes were serially washed in 1X TBST before being blocked with 1X
Blocking solution for 1 hour.
Table S.7
Tris-buffered saline with Tween-20 preparation.
Tris-HCl
NaCl
Tween-20
ddH2O
Table S.8
1X TBST
6.057 g
8.766 g
0.5 ml
1L
Blocking solution preparation.
1X Blocking solution
BSA (or Blotto)
2.5 g
Tween-20
200 µl
TBST
50 ml
Finally, membranes are probed with primary antibodies at their respective concentrations
diluted in 5ml of 1X TBST. Primary antibodies were incubated at 4°C overnight. Serial
washing followed by secondary antibody incubation at room temperature for 1 hour follows.
129
B.3
Antibodies Specificities
Table S.9
Antibody preparation.
Ang1
Concentration
1:250
Type
Primary, polyclonal, rabbit
Ang2
1:500
Primary, polyclonal, rabbit
β-actin
Goat anti-rabbit IgG
Chicken anti-mouse IgG
1:10000
1:10000
1:10000
Primary, monoclonal, mouse
Secondary
Secondary
Manufacturer, Cat #
Abcam, ab8451
Abcam,
ab65835
ICN, #691002
Cedarlane
Cedarlane
130