Journal of the Neurological Sciences 217 (2004) 125 – 130
`
`www.elsevier.com/locate/jns
`
`Review article
`
`Angiogenesis in multiple sclerosis: is it good, bad or an epiphenomenon?
`
`Shauna Kirk a, Joseph A. Frank b, Stephen Karlik a,c,d,*
`
`a Department of Pathology, University of Western Ontario, London, Ontario, Canada
`b Experimental Neuroimaging Section, Laboratory of Diagnostic Radiology Research, Clinical Center, National Institutes of Health, Bethesda, MD, USA
`c Department of Diagnostic Radiology, University of Western Ontario, London, Ontario, Canada
`d Department of Physiology, University of Western Ontario, London, Ontario, Canada
`
`Received 23 June 2003; received in revised form 20 October 2003; accepted 24 October 2003
`
`Abstract
`
`Characteristic pathological features of multiple sclerosis (MS) include inflammation, demyelination and axonal and oligodendrocyte loss.
`In addition, lesions can also have a significant vascular component. In this review, morphological, biochemical and radiological evidence is
`presented suggesting angiogenesis as a potential focus for investigation in MS. We hypothesize that angiogenesis plays a significant role in
`the MS lesion, perpetuating disease progression. Thus, treatment strategies that inhibit angiogenesis may decrease clinical and pathological
`signs of disease. Several approaches for testing this hypothesis are outlined.
`D 2003 Elsevier B.V. All rights reserved.
`
`Keywords: Angiogenesis; Multiple sclerosis; Experimental allergic encephalomyelitis; Neuroinflammation; VEGF; CNS pathology
`
`1. Introduction
`
`Multiple sclerosis (MS) is an inflammatory demyelinat-
`ing disease of the central nervous system (CNS) affecting
`approximately 350,000 individuals in North America alone
`[1]. Despite strong research efforts, the cause of MS remains
`elusive, the pathological mechanisms are not fully under-
`stood and the clinical course is highly variable, explaining
`why treatment options are still very limited. Without an
`established etiology or pathophysiology, the selection of
`treatments has concentrated almost exclusively on modify-
`ing the immune response. Research in MS concentrates on
`inflammatory changes, demyelination, axonal and oligoden-
`drocyte loss, and other characteristic pathological features of
`the MS plaque [2]; however, lesions also have a significant
`vascular component
`that warrants further investigation.
`Angiogenesis is the process leading to the development of
`new blood vessels from pre-existing ones [3,4]. An angio-
`genic response has been found to cause or contribute to an
`
`* Corresponding author. Diagnostic Imaging Room, 2MR21, London
`Health Sciences Center-University Campus, 339 Windermere Road,
`London, Ontario, Canada N6A 5A5. Tel.: +1-519-663-3648; fax: +1-519-
`663-3544.
`E-mail address: skarlik@uwo.ca (S. Karlik).
`
`0022-510X/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
`doi:10.1016/j.jns.2003.10.016
`
`increasing number of pathological conditions [5,6]. Al-
`though angiogenesis and other forms of inflammation ex-
`hibit a positive feedback relationship [3],
`the role of
`angiogenesis (rather than altered existing vessels) in the
`pathogenesis of MS and its potential as a therapeutic target
`has not yet been explored.
`
`2. Hypothesis
`
`We propose that the association between MS lesions and
`neovascularization is not merely an epiphenomenon, but
`represents a pathological mechanism contributing directly to
`sustained disease. It is our hypothesis that angiogenesis
`represents a significant component of the MS lesion con-
`tributing to disease progression, thus inhibition of angio-
`genesis may suppress pathological changes, ameliorating
`clinical signs of disease.
`
`3. Current knowledge
`
`Angiogenesis is generally quiescent in adults with the
`exception of certain tightly controlled physiological situa-
`tions such as female reproductive functions and tissue
`
`Apotex v. Novartis
`IPR2017-00854
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`
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`

`126
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`S. Kirk et al. / Journal of the Neurological Sciences 217 (2004) 125–130
`
`regeneration and repair [4]. An increase in vascularity acts to
`return the body to normal homeostasis by providing the
`necessary oxygen, nutrients and waste removal [7]. Whether
`or not angiogenesis occurs in a particular tissue depends on
`the balance between the relative amounts of pro- and anti-
`angiogenic factors [8]. Although the exact mechanism con-
`trolling angiogenesis in the CNS is not yet fully understood,
`many of the factors driving angiogenesis in other tissue are
`present during hypoxia/ischemia and tumor induced angio-
`genesis in the CNS [9 – 11]. Angiogenesis has been shown in
`an increasing number of pathological conditions including
`cancer, ischemic disease, diabetic retinopathy, blindness and
`inflammatory diseases (such as rheumatoid arthritis and
`psoriasis) [5,6]. While in some situations, such as cerebral
`and cardiovascular ischemic disease, angiogenesis is bene-
`ficial [9]; in many cases, such as rheumatoid arthritis,
`psoriasis and diabetic retinopathy, this increase in vascularity
`has been shown to be detrimental, leading to further damage
`[12,13].
`A relationship between blood vessels and MS lesions has
`been recognized for over 130 years. Edward Rindfleish [14]
`identified changes in both large and small vessels in the
`spinal cord and commented that all MS lesions were
`associated with abnormal blood vessels. MS lesions are
`typically centered on one or more veins in the white matter
`with particular association with the watershed veins of the
`periventricular white matter. Finger-like projections (Daw-
`son’s fingers) often extend from lesions and directly follow
`the course of the veins or venules [15 – 17]. Dawson’s
`fingers are thought to represent the invasion of the demy-
`
`elinating process into normal white matter [15]. Blood
`vessels are also associated with three of the four recently
`described MS lesion pathology classifications [18]. Pattern
`III, found primarily in acute disease, was not centered on
`veins and venules. This pattern has been shown to be
`associated with hypoxia-inducible factor 1 (HIF-1) [19],
`essential to hypoxia induced, vascular endothelial growth
`factor (VEGF) mediated angiogenesis [20,21], suggesting
`pattern III lesions may progress to a more typical perivas-
`cular pattern. As cells must be within 100 – 200 Am of a
`blood vessel to survive [22,23], MS lesions would require
`sufficient vascularization to permit delivery of additional
`hematogenous cells to maintain the inflammatory state.
`Several key components in the pathophysiology of MS
`(Fig. 1) are also associated with angiogenesis. Matrix metal-
`loproteases (MMP)-1, -2, -3 and -9, intercellular cell adhe-
`sion molecule (ICAM)-1, vascular cell adhesion molecule
`(VCAM)-1 and E-selectin facilitate in the entry of mono-
`nuclear cells through the blood – brain barrier (BBB) in MS
`[16,24] and are involved with the breakdown of the base-
`ment membrane in angiogenesis. This breakdown results in
`the further release of growth factors and angiogenic signal-
`ing molecules [25]. Inflammatory mediators,
`interferon
`(IFN)-g and tumor necrosis factor (TNF)-a/-h, are promi-
`nent in MS [16] and are angiogenic regulators [26]. In MS,
`elevated nitric oxide (NO) levels correlate well with clinical
`and magnetic resonance (MR) markers of disease progres-
`sion [27]. NO has been found to contribute both directly and
`indirectly to angiogenesis in inflammatory, vascular diseases
`and tumor expansion [28]. Endothelin-1 (ET-1), which
`
`Fig. 1. Potential role for angiogenesis in MS. (1) Initially immune cells bind to the BBB through a variety of cell adhesion molecules. (2) Cells infiltrate the
`perivascular space. (3) Cytokine, immune and antibody mediated attack on the myelin in the perivascular space. (4) Infiltration of immune cells into the
`parenchyma. (5) Release of inflammatory and hypoxic mediators including VEGF, HIF-1, NO, ET-1 from various cell types. (6) Angiogenic signals cause the
`initiation of the formation of new blood vessels into the lesion area.
`
`

`

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`127
`
`stimulates neovascularization in concert with VEGF, corre-
`lates with tumor vascularity and malignancy, and induces
`MMP-2 production [29], has also been reported to be
`significantly elevated in MS patients [30]. VEGF, an un-
`equivocal angiogenic factor that signals the proliferation and
`migration of endothelial cells in angiogenesis [8,31], has
`recently been reported in both MS and its animal model
`experimental allergic encephalomyelitis (EAE) plaques
`[32]. In EAE induced rats an intracerebral infusion of VEGF
`resulted in an inflammatory response in the brain which was
`not found in vehicle-infused animals [32]. VEGF acts both
`directly and indirectly to promote angiogenesis, contributing
`directly to angiogenesis by acting as a specific mitogen and
`potent chemo-attractant
`to endothelial cells, as well as
`enhancing vascular permeability [33]. Indirectly, VEGF
`induces endothelial cells to release other factors, such as
`MMPs and ICAM-1, involved in the angiogenic process
`[33,34]. Several other angiogenic cytokines act at least in
`part by up-regulating VEGF expression [35].
`Further evidence for altered vascularity in MS is the
`existence of ‘‘ring enhancement’’ in contrast enhanced MRI
`at the periphery, but not at the center of chronic lesions [36].
`Early enhancing nodular lesions progressed to ring enhance-
`ment growing in size over time, supporting the belief that
`enhancement and possibly demyelination occurred from the
`center outwards [37]. The radiological evidence of perme-
`ability changes in MS has been assumed to represent the
`increased leakiness of existing vessels produced by inflam-
`matory modulators [38]. However, the evidence is equally
`indicative of new vessels and expansion of the regional blood
`volume, wherein the newly formed vessels are permeable to
`gadolinium-DTPA (Gd). Increased permeability in newly
`formed vessels could also be the cause of the prolonged T1
`and T2 seen in MS lesions or altered magnetization transfer
`ratio and diffusion weighted imaging observed in normal
`appearing white matter (NAWM).
`Rashid et al. [39] showed an increase in cerebral perfu-
`sion in relapsing-remitting (RR-) and secondary-progressive
`(SP-) MS patients compared to controls, which may provide
`evidence that there is an increase in the number of vessels in
`these inflammatory lesions. Recently, a serial MRI study of
`Gd enhancing MS lesions for local perfusion measurement
`found a significant increase in cerebral blood volume and
`cerebral blood flow, not only at the time of initial Gd
`enhancement compared to baseline, but also as early as
`three weeks prior to enhancement [40]. This indicates a
`significant role for the vasculature in NAWM very early on
`in lesion formation.
`In addition to an up-regulation of VEGF expression in
`both acute and chronic plaques, Proescholtd et al. [32] also
`demonstrated that blood vessels within lesions showed an
`irregular morphology consistent with angiogenesis. In acute
`MS lesions Ludwin et al. [41] reported an increase in the
`number and size of blood vessels with increases in endo-
`thelial cell number and mitotic count. Neovascularization
`was marked within, and at the edge of MS lesions, but also
`
`extended into the surrounding peri-plaque, often highlight-
`ing areas of acute inflammation or reactive gliosis. Ring
`enhancing lesions on MRI demonstrated increased vascu-
`larity and angiogenesis around the peripheral aspects of the
`lesion. VEGF and HIF were also found to be elevated in MS
`brains compared to normal tissues. In addition, chronic MS
`lesions appeared to have an increase in the number of
`vessels within a lesion although exact quantification was
`difficult due to scarring and shrinkage of the tissues [41].
`This is the first study in MS to demonstrate an increase in
`the number of blood vessels, however, the specific role of
`angiogenesis in disease progression and in treatment has not
`yet been evaluated.
`
`4. Angiogenesis as a therapeutic target in MS
`
`Many diseases are driven by persistent unregulated
`angiogenesis providing an opportunity for therapeutic in-
`tervention [12]. As new blood vessels are known to play a
`role both in the continued pathogenesis of chronic inflam-
`mation and in wound healing and repair processes, the issue
`of therapeutic intervention in angiogenesis remains contro-
`versial. An important consideration with anti-angiogenic
`therapy is the potential for impaired healing [42]. There is
`evidence to suggest that repair and regeneration can occur
`in the absence of angiogenesis. An anti-angiogenic therapy,
`CM101, was beneficial in the treatment of an experimental
`model of spinal cord injury. In this model, the inhibition of
`angiogenesis minimized both acute and chronic inflamma-
`tion in the CNS leading to the recovery of walking ability
`in the majority of treated mice [43]. In a study of cutaneous
`wound healing, CM101 was found to stimulate wound
`healing. The overall number of blood vessels was decreased
`and the inflammatory process was inhibited, reducing
`infiltration of activated leukocytes thus, preventing further
`tissue damage [44]. A study of dermal wound repair
`showed inhibition of angiogenesis though an alpha-v integ-
`rin blocking antibody, had no detrimental effects on wound
`repair [45]. When an avh3 antagonist was used in the
`treatment of an experimental model of rheumatoid arthritis
`it was shown to be effective not only in acute disease, but
`also resulted in the apoptosis of angiogenic blood vessels
`and effectively diminished clinical and pathological signs of
`disease [46].
`The positive feedback relationship between inflammation
`and angiogenesis can be advantageous under some circum-
`stances, however, as MS is an autoimmune disorder, per-
`sistent
`inflammation against a self antigen results in
`destruction of normal tissue. The potential advantages of
`inhibiting angiogenesis include halting the supply of
`nutrients and further inflammatory cells into the inflamed
`tissue and inhibiting the production of endothelial cell
`derived soluble factors [23]. The benefit of inhibiting
`angiogenesis has been seen in the animal models and early
`clinical studies of other autoimmune disorders such as
`
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`

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`
`rheumatoid arthritis [46 – 50], diabetic retinopathy [51 – 54]
`and psoriasis [55]. Although angiogenesis is not likely the
`primary event
`in the pathogenesis of MS,
`its role in
`continued disease progression makes it an important target
`for therapeutic intervention.
`Most, if not all, of the treatments currently used in MS
`display anti-angiogenic properties in other diseases as
`shown by in vivo or in vitro studies. In MS, IFN-h reduces
`the proliferation of T cells, decreases the production of
`TNFa and antigen presentation and alters cytokine produc-
`tion to favor a Th2 pattern [56]. IFN-h also reduces the
`passage of immune cells across the BBB by affecting
`adhesion molecules, chemokines and proteases [16]. IFNs
`in general, are multi-functional cytokines shown to have
`anti-angiogenic properties shown both in vivo and in vitro
`and are used to treat tumor angiogenesis [57,58].
`Corticosteroids have been found beneficial in reducing
`the length of significant clinical relapses in some patients
`with RR-MS [59]. They have potent anti-inflammatory and
`immunosuppressive properties as well as having a stabiliz-
`ing effect on the BBB [60]. Certain corticosteroids have
`been found to be angiostatic, inhibiting the development of
`new blood vessels in tumors [61] and chronic inflammation
`[62,63].
`Immunosuppressive therapies (i.e. mitoxantrone, cyclo-
`phosphamide) are used in approximately 10% of MS
`patients to slow the progression of disease in RR-MS
`patients [64]. Immunosuppressive therapies are commonly
`used in the treatment of cancer and have been found to be
`highly anti-angiogenic [65,66].
`
`5. Hypothesis testing
`
`Clinical, pathological and imaging studies can be uti-
`lized to determine the role of angiogenesis and its potential
`as a therapeutic target
`in MS. EAE can be induced in
`several strains of rodents and non-human primates resulting
`in disease ranging from an acute attack with little or no
`demyelination to a chronic progressive disorder with ex-
`tensive demyelination and some remyelination [67,1].
`Studies of EAE using immunohistochemistry and histology
`can be used to evaluate the temporal relationship of
`angiogenic markers, blood vessel formation and clinical
`and pathological disease progression. Therapeutic studies
`in EAE can be utilized to test both anti-angiogenic and
`angiogenesis inducing agents. Inhibition of angiogenesis
`has the potential to also alleviate chronic inflammation [6].
`By suppressing the development of neovascularization,
`nutrients and new inflammatory cells will not reach the
`site of inflammation. This will prevent endothelial cell
`activation, proliferation and vascular remodeling, inhibiting
`the production of endothelial cell derived factors such as
`MMPs, VEGF receptors and cytokines [23]. Therapy that
`induces angiogenesis can help to determine if an increase
`in vascularity contributes to accelerated disease progres-
`
`sion. Conversely, some evidence suggests revascularization
`is necessary for promoting neural repair and regeneration,
`and prevention of the formation of new blood vessels could
`be detrimental [68]. EAE offers an opportunity to test our
`belief that
`tissue repair cannot be initiated until further
`damage has been halted, potentially requiring the resolu-
`tion of angiogenesis.
`Studies assessing the clinical signs and circulating an-
`giogenic markers (e.g. VEGF, bFGF) of both treated and
`untreated MS patients over time may be used as biomarkers
`to determine if current therapies exhibit an anti-angiogenic
`effect on MS patients and if so, if there is clinical benefit to
`decreased angiogenesis.
`Randomized clinical trials using anti-angiogenic agents
`either currently in use or in clinical trials for cancer and
`arthritis may provide additional therapeutic options for MS
`patients. Therapeutic strategies to inhibit angiogenesis target
`various aspects such as preventing matrix breakdown,
`inhibiting endothelial cell proliferation,
`interfering with
`specific activators of angiogenesis and inhibiting endothelial
`cell survival signaling. There are several ongoing clinical
`trials of anti-angiogenic therapies [69], however, to date, the
`studies have met varying degrees of success [70]. As
`knowledge of the biochemical and molecular mechanisms
`involved in angiogenesis increases, promising new thera-
`peutic strategies will offer more focused treatment leading to
`new therapeutic trials.
`Certain medications used for other indications have been
`shown to have anti-angiogenic properties. Minocycline
`hydrochloride used in the long-term treatment of acne
`patients has been effective in two different models of EAE
`[71,72] and is a potent inhibitor of angiogenesis in tumors
`[73] and in vitro [74,75]. A retrospective study of the
`disease course of individuals with MS concurrently receiv-
`ing long-term therapy with minocyline for acne may provide
`additional information into the benefit of inhibiting angio-
`genesis in MS.
`Clinical and EAE imaging studies could be utilized to
`determine the extent to which neovascularization contrib-
`utes to MRI findings and establish the clinical relevance of
`these findings.
`
`6. Summary and hypothesis
`
`MS is an inflammatory, demyelinating disorder of the
`CNS with vascular features. A firm link has been estab-
`lished between chronic inflammation and angiogenesis and
`evidence in MS is mounting to suggest a significant role
`for neovascularization in the progression of disease. Stud-
`ies of the role of angiogenesis over the course of MS and
`EAE have the potential to provide evidence concerning a
`key mechanism in the progression of disease. The evolu-
`tion and spread of lesions facilitated by angiogenesis may
`present a potential target for therapeutic intervention for
`MS.
`
`

`

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`129
`
`Acknowledgements
`
`Supported by the MS Society of Canada.
`
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