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Characterizing the expression profile of angiogenic proteins after acute spinal cord injury Ng, Tsz Lui Michelle 2012

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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  79  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  80  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  81  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  82  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  83  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.  84  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  85  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.  86  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  87  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  88  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.  89  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 90  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  91  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  92  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.  93  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  94  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  95  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].  96  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  97  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)  98  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  Bibliography 1. 2. 3.  4. 5. 6.  7. 8. 9. 10.  11.  12.  13. 14.  15.  16.  17. 18.  Farry, A., Baxter, David, The Incidence and Prevalence of Spinal Cord Injury in Canada. 2010, Rick Hansen Institute and Urban Futures Institute: Vancouver, BC. Spinal Cord Injury Fact and Figures at a Glance. 2011, National Spinal Cord Injury Statistical Center. Noonan, V.K.F., M. ; Farry, A. ; Baxter, D. ; Singh, A. ; Fehlings, M.G. ; Dvorak, M.F. , Incidence and Prevalence of Spinal Cord Injury in Canada: A National Perspective. 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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  

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