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Research report

Expression of vascular endothelial growth factor by reactive astrocytes

and associated neoangiogenesis

a a,b a a c

Bodour Salhia , Lilyana Angelov

, Luba Roncari , Xiaoli Wu , Patrick Shannon ,

a,b ,

_*

Abhijit Guha

Labatts Brain Tumor Center, Hospital for Sick Children, Toronto, Ontario, Canada b

Division of Neurosurgery, University Health Network, University of Toronto, Western Division, Toronto, Ontario, Canada c

Division of Neuropathology, University Health Network, University of Toronto, Toronto, Ontario, Canada Accepted 8 August 2000

Abstract

Injury to the central nervous system (CNS) invokes a reparative response known as astrogliosis, characterized largely by hypertrophy, proliferation and increased expression of glial fibrillary acidic protein (GFAP), resulting in reactive astrocytosis. Based on our prior observation that peritumoral reactive astrocytes express Vascular Endothelial Growth Factor (VEGF), a highly potent and specific angiogenic growth factor, we have hypothesized that reactive astrocytosis also contributes to the neovascularization associated with astrogliosis. To evaluate this hypothesis we evaluated human surgical / autopsy specimens from a variety of CNS disorders that induce astrogliosis and an experimental CNS needle injury model in wild type and GFAP:Green Fluorescent Protein (GFP) transgenic mice. Using computer image semi-quantitative analysis we evaluated the number of GFAP-positive reactive astrocytes, degree of VEGF expression by these astrocytes, associated Factor VIII-positive microvascular density (MVD) and Ki-67 proliferating endothelial cells. The degree of reactive astrocytosis correlated to levels of VEGF immunoreactivity and MVD in the neuropathological specimens. The mouse–needle–stick brain injury model demonstrated this correlation was temporally and spatially related and maximal after 1 week. These results, involving both human pathology specimens augmented by experimental animal data, supports our hypothesis that the neoangiogenesis associated with reactive astrogliosis is correlated to increased reactive astrocytosis and associated VEGF expression.

Theme: Cellular and molecular biology

Topic: Neuroglia

Keywords: Astrogliosis; GFAP; Neoangiogenesis; Microvascular density; Reactive astrocytes; VEGF

1. Introduction cell processes and bodies with increased expression of the astrocyte specific cytoskeletal intermediate filament Glial Reactive astrogliosis is a normal reparative mechanism Fibrillary Acidic Protein(GFAP) [7,11,14,35,41,50,64,65]. induced in many CNS disease processes such as ischemia, This reactive astrocytic response has been noted to occur infection, trauma, degenerative diseases, epilepsy and brain within 2 days after experimental brain injury in rodents tumors [29,31,49,50,63]. Amongst the various cellular and approximately 1 week in humans and may persist for responses of reactive gliosis, there is a prominent as- several weeks with progressive decline over months to trocytic response [31,58] comprised of normally quiescent years [43,47,50,63]. The function of this florid reactive astrocytes proliferating, undergoing hypertrophy of their astrocytosis is not fully deciphered, but is postulated to play a role in the healing phase after insult to the CNS by monitoring and controlling the molecular and ionic

con-*Corresponding author. Division of Neurosurgery, University Health _{tents of the CNS extracellular space. This is likely} Network, University of Toronto, Western Division, 399 Bathurst Street,

achieved via multiple membrane receptors for

neurotrans-Toronto, Ontario, Canada M5T 2S8. Tel.:11-416-603-5740; fax: 1

1-mitter peptides, growth factors and specific ion channels

416-603-5298.

E-mail address: [email protected] (A. Guha). that are known to be expressed by normal and reactive

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astrocytes [31,50,63]. However, it is hypothesized that trocytosis, neoangiogenesis and VEGF expression from a even moderate reactive astrocytosis may interfere with spectrum of conditions including Alzheimer’s disease, eventual reconnection of functional neuronal circuitry by brain abscess, peritumoral brain from metastatic car-inhibiting axonal regeneration, preventing remyelination or cinoma, head injury and cerebral infarcts. Further, to promoting abnormal neuronal connections with increased evaluate the temporal relationship of reactive astrocytosis

seizure foci [57]. and neoangiogenesis, a stereotactic brain injury model was

There is currently little understanding of the molecular utilized. In order to obtain the proliferative index ([ _Ki67 mechanisms regulating reactive astrocytosis. Several ob- positive cells / 1000 endothelial cells3100) of endothelial servations suggest that reactive astrocytosis is a regulated cells, surgical specimens from brain abscess and metastatic process involving cytokines, inflammatory mediators, carcinoma were stained for Ki-67. In addition, a CNS growth factors and physiological stimuli such as hypoxia injury model was utilized in transgenic mice which express [45,50,69]. For example, axonal growth and regeneration Green Fluorescent Protein (GFP) under the control of the after trauma and cerebral infarction are retarded by inhib- astrocyte specific GFAP promoter [5], to co-localize VEGF itory proteins associated with the intense astrogliotic scar expression in reactive astrocytes.

[6,45,50]. Second, reactive astrocytosis although most intense immediately surrounding the area of injury, is

found at much removed distances suggestive of soluble 2. Methods

trophic factors acting in a paracrine fashion [50]. While a

variety of these trophic factors have been studied in 2.1. Evaluation of human neuropathological diseases astrocyte cell culture experiments, their role and

interac-tions in the astrogliotic response in vivo is poorly under- In order to evaluate neoangiogenesis and VEGF expres-stood. Specifically, polypeptide growth factors such as sion in a variety of reactive astrogliotic specimens, 23 basic Fibroblast Growth Factor (bFGF), Epidermal Growth cases of neurosurgical and autopsy specimens obtained Factor (EGF), Transforming Growth Factor (TGF-a), from our neuropathology department were examined. Platelet Derived Growth Factor (PDGF), Cilliary Neuro- These cases represented a spectrum of benign and malig-trophic Factor (CNTF) and cytokines such as Interleukin-1 nant diseases that are known to induce reactive astrocytosis (IL1) and Tumor Necrosis Factor (TNF-a), have all been in the surrounding brain and included: four Alzheimer’s implicated in the astrogliotic response [3,13,32,37, Disease (AD), three bacterial abscesses, five carcinomas,

49,50,59,68,69]. three head injuries and seven infarcts (two acute:,7 days,

Part of any reparative tissue process is neoangiogenesis, two subacute: 7–30 days, three old: .30 days). Normal vividly demonstrated in the collagen fibrovascular response autopsy brain tissue was used as a control. Standard associated with wound healing and one that can be hematoxylin and eosin (H&E) staining was performed on speculated to be an important component of reactive 5-mm tissue sections from paraffin embedded tissue astrocytosis in the CNS [10]. Amongst the regulators of blocks, which were independently reviewed by a neuro-neoangiogenesis, Vascular Endothelial Growth Factor pathologist (PS). Sequential sections were incubated over-(VEGF) is highly potent and specific by activating its night at 48C with a primary rabbit antibody against GFAP cognate receptors on the endothelial cells resulting in their (1:3000, Dako Corp, CA) to mark reactive astrocytes, a proliferation, migration and sprouting [17,27,61]. VEGF human anti-Factor VIII antibody (1:2000, Dako Corp) to has been implicated in most physiological and pathological determine the microvascular density (MVD) or a polyclon-processes involving neovascularization including embryo- al rabbit anti-VEGF antibody (1:50, Santa Cruz Biotech-genesis, wound healing, tumor growth, myocardial is- nology Inc., CA) that recognizes all four VEGF isoforms. chemia, ocular neovascular diseases and chronic diseases Detection was through the ABC (Vector)-DAB (VectaStain such as rheumatoid arthritis [16,21]. In addition to its Elite, CA) system. The sections were analyzed using the angiogenic effects, VEGF is also a potent edemogenic MicroComputer Image Device (MCID; Imaging Research agent, resulting in disruption of the blood–brain-barrier Inc., Canada), linked to a color CCD camera (Sony DXC (BBB), and hence was also known as Vascular Permeabili- 970 MD) mounted on a transmitted-light microscope ty Factor (VPF). We observed that VEGF / VPF was co- (Zeiss Axioskop) fitted with a Ludl Biopoint motorized expressed with GFAP-positive reactive astrocytes in the stage. Microscope fields (4003magnification) were ac-peritumoral edematous brain around meningiomas, where quired and digitized for each of the sequential sections its expression was correlated to the vascularity and associ- stained for GFAP, Factor VIII and VEGF, with 4–6 high-ated edema induced by these CNS tumors [56]. This powered fields (HPF) analyzed in each tissue section. suggested that reactive astrocytes may express VEGF, These HPFs were chosen to be adjacent to the site of tissue modulating neoangiogenesis and also the edema and break necrosis by the underlying pathology (i.e, adjacent to the down of the blood brain barrier associated with reactive abscess wall, carcinoma, infarct, contusion) or in the astrogliosis. To address this hypothesis, immunohisto- mesial temporal lobes of the AD specimens. All raw values chemical techniques were used to quantify reactive as- were reported as a mean6standard error of the means

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(S.E.M.) and also represented as a percentage of the endogenous GFAP was performed by incubating with a maximum value obtained. The extent of astrocytosis was rabbit anti-GFAP antibody (1:10, Dako) or mouse mono-assessed by averaging the number of GFAP-positive clonal anti-VEGF antibody (1:10, UBI) for 1 h at room reactive astrocytes / 4–6 high-powered fields (HPF). MVD temperature. After three washes in PBS containing 1% counts were derived by averaging the number of Factor BSA and 0.9% NaCl, the sections were incubated with VIII-positive vessels / 4–6 HPF and VEGF staining was either Cy5-conjugated goat anti-mouse IgG (1:50, Jackson quantified by averaging the percentage VEGF immuno- ImmunoResearch Laboratories Inc.) or Cy3-conjugated

reactivity / 4–6 HPF. goat anti-rabbit IgG (1:20, Jackson ImmunoResearch

In three cases of brain abscess and two cases of Laboratories Inc.) for 1 h at room temperature. Sections metastatic carcinoma, sections were stained for Ki-67 were washed in PBS and mounted for immunofluorescent (MIB-1, 1:50, Immunotech) after microwave antigen re- microscopy. Sections were evaluated with the green trieval and the proliferative index of the endothelium was immunofluorescence filter to detect expression of the GFP obtained. The proliferative index was determined by transgene under the GFAP promoter and for either endog-counting 1000 endothelial cell nuclei and determining the enous GFAP (Cy3) or VEGF (Cy5).

percentage staining positive for Ki-67.

2.2. Wild type and transgenic GFAP:GFP mouse 3. Results

stereotactic needle brain injury

3.1. Reactive astrocytosis, VEGF immunoreactivity and To determine the temporal association between reactive neoangiogenesis in human CNS diseases

astrocytosis, neoangiogenesis and VEGF expression, a

reproducible stereotactic needle stick injury model to the The mean age of patients with the different diseases frontal cerebral cortex of mice was used [42,47,50]. A total were 79 years — AD; 38 years — Abscess; 65 years — of 27 CD1 mice were anesthetized with 0.5 ml of 2.5% Peri-metastatic carcinoma brain; 52 years — Head Injury; Avertin (2,2,2-tribromoethanol and 2-methyl-2-butanol) 71 years — Cerebral infarcts. Reactive astrocytosis with (Sigma-Aldrich Chemical Company Inc., WI) by intra- increased numbers of large and darkly stained GFAP-peritoneal (i.p.) injection. The head was stabilized in a positive reactive astrocytes were a prominent feature of all stereotactic frame and a burr hole drilled 2 mm anterior to these disease processes compared to normal brain, al-the coronal suture and 2 mm lateral to al-the saggital suture. though the degree of reactive astrocytosis varied with the The needle of a 5-ml Hamilton syringe was then inserted type of pathology, as demonstrated in Table 1, Fig. 1. The through the burr hole to a depth of 2.5 mm for a duration most exuberant reactive astrocytosis was induced in the of 1 min, then withdrawn. The contra-lateral frontal lobe brain surrounding bacterial abscesses (Table 1, Fig. 1-Row was used as the non-injured control side in all evaluations. A), with an average raw value of 82628 GFAP-positive The track was made in cortical gray matter, not penetrating astrocytes / HPF. In comparison, diffuse head injury (Fig. sub-cortical areas that normally contain higher numbers of 1-Row C), induced the lowest amount of reactive as-GFAP-positive cells [8]. All mice tolerated the procedure trocytosis, with a raw value 2067 GFAP-positive as-well. Three mice were sacrificed on each of days 1–9 trocytes / HPF, though this result should be tempered due to post-injury by cervical dislocation and the brains fixed in our lack of knowledge of severity or the time of the formalin for 24 h and embedded in paraffin for histological autopsy after the head injury. The amount of VEGF processing. Axial, 6-mm thick paraffin sections at right immunoreactivity paralleled the degree of induced reactive angles to the needle tract were cut, stained with anti-GFAP, astrocytosis, except for cerebral infarcts and peri-tumoral anti-Factor VIII and anti-VEGF antibodies and analyzed brain, where VEGF immunoreactivity was disproportion-with the computer assisted image analysis system as ately higher (Table 1). The number of Factor VIII-positive

above. blood vessels (MVD), as a measure of neoangiogenesis,

Three FVB / N background GFAP:GFP transgenic mice was related to the degree of reactive astrocytosis and (a gift from Dr. A. Messing, Madison, WI, USA) under- VEGF immunoreactivity. This is best exemplified by went a similar needle tract injury and were evaluated 7 MVD, raw counts of 2063 vessels / HPF, associated with days after injury with immunofluorescence microscopy. the florid reactive astrocytosis in bacterial abscess, com-Mice were sacrificed by cervical dislocation. Brains were pared to only 661 vessels / HPF in brains with prior head removed within 5 min of sacrifice, embedded in OCT injury, AD or infarcts. In these latter conditions, the degree (Tissue Tek) embedding medium on dry ice and stored at of reactive astrocytosis and associated neoangiogenesis

2708C until further processing. Axial cryostat sections although relatively low compared to brain around bacterial were fixed in ice cold acetone for 3 min at2208C, washed abscess, was still significantly high relative to normal brain with PBS for 10 min and blocked in a solution of PBS (Table 1). In all cases, astrocytosis, VEGF immunoreactivi-containing 1% BSA and 0.9% NaCl for 30 min at room ty and MVD was increased from normal brain (Fig. 1-Row temperature. Double immunofluourescence for VEGF and F). In addition, blood vessels associated with brain abscess

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Table 1

a Semi-quantitative analysis of immunohistochemical staining in a variety of human neuropathologies

Diagnosis n Reactive VEGF Vascularity

Astrocytosis Expression (No. Factor

(No. GFAP- (VEGF VIII-stained

Positive immunoreactivity vessels / HPF)

Cells / HPF) / HPF)

Normal 1 261 (2) 461 (9) 261 (10)

Alzheimer’s disease 4 3962 (48) 2564 (54) 661 (30)

Bacterial abscess 3 82628 (100) 661 (100) 2063 (100)

Head injury 3 2067 (24) 1364 (28) 661 (30)

Infarct 6 2263 (27) 2363 (50) 661 (30)

Peritumoral brain 4 2861 (34) 3061 (65) 1163 (55)

Reactive astrocytosis, VEGF expression and vascularity in a spectrum of human neuropathological specimens. Raw mean values6S.E.M. are shown and values are reported as a percentage of the maximum in brackets. All slides were quantified at 4003magnification. VEGF, vascular endothelial growth factor; S.E.M., standard error of the mean; GFAP, glial fibrillary acidic protein; HPF, High Power Field.

appeared larger and more robust than other CNS diseases parameters subsequently decreased towards normal levels investigated and the blood vessels associated with head by day 9. While the peak days for each parameter analyzed injury seem much smaller compared to other conditions. In do not occur on the same day in this experimental the astrogliotic response associated with AD and cerebral paradigm, the overall temporal correlation between them is infarcts, the vascularity, although increased from normal clearly demonstrated. The GFAP:GFP transgenic mice brain, was relatively less compared to the degree of VEGF were used for co-localization experiments in a needle stick immunoreactivity by reactive astrocytes. The proliferative injury model to confirm that the reactive astrocytes in-index of endothelial cells ranged from 8 to 12% in brain duced by the needle tract injury (* in Fig. 4) were abscesses (n53) and from 2 to 6% (n52) near metastatic expressing VEGF. Green immunofluorescence denoted the tumor. These results clearly demonstrate active neoan- expression of the GFP transgene under regulation of the giogenesis in these two conditions. Material taken from GFAP promoter (Fig. 4A). These same GFP expressing autopsy specimens was not used to determine proliferative cells were reactive astrocytes, as they also expressed index because, in our experience, detection of this antigen endogenous GFAP as detected by double immunofluores-is not reliable in timmunofluores-issue undergoing prolonged fixation. cence, where the cells are yellow due to the combination of green and the Cy3 secondary antibody used to detect 3.2. Temporal and spatial expression of reactive endogenous GFAP (Fig. 4B). On adjacent sections, the astrocytosis, VEGF immunoreactivity and GFP reactive astrocytes are the same cells that are express-neoangiogenesis ing VEGF, where they appear red due to combination of green and Cy5 immunofluorescence used to detect VEGF The mouse stereotactic needle stick injury resulted in a (Fig. 4C). There were no significant reactive GFP-positive distinct tract that could be consistently visualized on the astrocytes in the non-injured contra-lateral hemisphere injured side with a surrounding reactive astrogliotic re- (Fig. 4D).

sponse (Fig. 2A). The injury induced GFAP-positive astrocytes (Fig. 2B) to express VEGF (Fig. 2C) with

accompanying neoangiogenesis, as marked by Factor VIII- 4. Discussion

positive endothelial cells (Fig. 2D). The number of

GFAP-positive reactive astrocytes reached a maximum on day-7 Astrogliosis and the formation of the glial scar, with a post-injury, which was taken as 100% for comparison with predominant reactive astrocytosis component, is an im-the oim-ther time points analyzed (Figs. 2, 3). VEGF immuno- portant and widespread neuropathological response to reactivity increased to its maximum level (100%) on day 6 diverse neurological insults, a feature that is well con-post-injury, when the reactive astrocytic response had served across different species. This prominent, rapid and reached 47614% of maximal day-7 levels (Fig. 3). The evolutionarily conserved reparative response, suggests that number of Factor VIII-positive blood vessels (MVD) reactive astrocytosis fulfills an important function in the reached a maximal level (100%) on day 5 post-injury, CNS. However, the exact role of reactive astrocytosis and when the degree of reactive astrocytosis was 31614% and whether it is beneficial or ultimately detrimental to CNS VEGF immunoreactivity was 5967% of maximal levels recovery after injury remains unclear and controversial. on day 7 and day 6 respectively (Figs. 2, 3). Therefore, Reactive astrogliosis is a dynamic process that leads to a there was a progressive increase in reactive astrocytosis, densely interwoven glial scar composed of many cells VEGF immunoreactivity and neoangiogenesis after injury, including reactive astrocytes, microglia, macrophages and with maximal levels clustering around days 5–7. All three endothelial cells [12,24,26,31,53]. Reactive astrocytes

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Fig. 1. Astrocytosis and neoangiogenesis in human CNS disease. Photomicrographs (scale bar depicts 50mm in length) illustrating immunohistochemical staining of a spectrum of human neuropathological disease states with: Row A — Brain Abscess; Row B — Alzheimer’s Disease; Row C — Old Infarct; Row D — Peritumoral Brain; Row E — Head Injury; Row F — Normal Brain; Column 1 — anti-glial fibrillary acidic protein (GFAP) antibody; Column 2 — anti-vascular endothelial growth factor (VEGF) antibody; Column 3 — anti-Factor VIII antibody. When compared to the staining observed in normal brain, GFAP reactive astrocytes and neoangiogenesis as indicated by VEGF immunoreactivity and Factor VIII-stained vessels, is common and increased in all of the human pathologies (reddish / brown staining). GFAP positivity is noted in the reactive astrocytes and astrocytic foot processes around blood vessels. The most intense staining for all three parameters is observed in the tissue taken from the brain surrounding the abscess.

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Fig. 2. Visualization and characterization of mouse–needle–stick injury. Immunohistochemical sections of mouse brain stained for GFAP, VEGF and Factor VIII. (A) Coronal section through the injury site (scale bar depicts 2 mm in length) displaying GFAP immunoreactivity (brown staining) in reactive astrocytes straddling the tract site (arrow). The contralateral non-injured site (N) is GFAP negative in the same cortical region. Mouse brain sections at higher power magnification at the site of the needle stick injury (day 7 post-injury) (scale bar depicts 50mm in length) stained with (B) anti-glial fibrillary acidic protein (GFAP) antibody; (C) anti-vascular endothelial growth factor (VEGF) antibody; (D) anti-Factor VIII antibody. Day 7 post-injury is the maximum GFAP response, also shown in Fig. 3. The injury tract (*) can be seen in the upper left corner of each section. Arrows in B and C are pointing to positively stained reactive astrocytes.

form scars by extending their long, thick cytoplasmic neoangiogenesis (MVD) and VEGF immunoreactivity in a processes toward the site of the lesion [29]. It is likely that variety of human CNS pathologies (see Table 1, Fig. 1). the glial scar can serve to separate normal brain from the Reactive astrocytosis, neoangiogenesis and increased surrounding damaged tissue, but its formation may also be VEGF was maximal in response to bacterial brain abscess harmful by essentially creating a mechanical barrier [50] and probably reflects the acuteness of the illness and the which may impede neuronal dendritic growth, remodeling type of response that it induces in the CNS. In an abscess, and axonal regeneration [25,29,50,63,71]. In addition, the in response to the released toxins, there is an inflammatory glial scar may contribute to electrical instability in the cellular response, components of which themselves release region and in turn promote seizure activity [50]. However, VEGF and other inflammatory cytokines modulators. This more recent evidence suggests that astrogliosis may actual- leads not only to a vigorous reactive astrogliotic response ly facilitate CNS recovery through neurotrophic factor but it is also one of the few CNS diseases that induces a production around the area of the lesion [2,34,67]. In collagen fibrovascular response.

summary, like the common collagen fibrovascular wound The amount of VEGF immunoreactivity and MVD healing process, reactive astrogliosis serves a beneficial exceeded the degree of reactive astrocytosis in the brain CNS reparative function, however, under certain circum- around carcinomas (Table 1), perhaps attributable to the stances it may be detrimental much like exuberant scar and additive expression of VEGF from the GFAP positive

keloid formation in other tissues. reactive astrocytes and the carcinoma cells themselves

This study demonstrates, using human specimens and [22]. In cerebral infarcts, irrespective of whether it was experimental models of CNS injury, that reactive as- acute, subacute or old, VEGF immunoreactivity was higher trocytosis is accompanied by a neoangiogenic response, than the degree of reactive astrocytosis or neoangiogenic similar to the collagen fibrovascular response elsewhere. response (see Table 1). This may reflect the potent hypoxic Furthermore, we speculate based on our results that a induction of VEGF expression by the surviving reactive potential source of this angiogenic response lies in in- astrocytes [15], without a concomitant increase in the creased VEGF secretion by the quiescent reactive as- number of astrocytes or the ability to recruit new blood trocytes. This conclusion is based on the overall consistent vessels due to the cerebral ischemia. This data is similar to relationship between GFAP-positive reactive astrocytes, results in a study by Plate et al., where in a middle cerebral

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Fig. 3. Temporal association of astrocytosis, VEGF immunoreactivity and neoangiogenesis in the needle stick injury model. Histogram (depicting percentage of the maximum mean6S.E.M.) demonstrating the temporal association in the expression patterns of GFAP, VEGF and MVD following mouse–needle–stick injury. The maximal number of GFAP-positive astrocytes, VEGF expression and neoangiogenesis was temporally correlated and occurred between days 5–7 post-injury supporting a relationship in the time course of the astrogliotic and neoangiogenic responses.

artery occlusion in rats, VEGF mRNA expression was positive reactive astrocytes. Our results confirm previous consistently greater across different time points than the findings that reactive astrocytes after a stab wound injury number of CD31 stained vessels [54]. In neurodegenera- are VEGF positive [46,52] and also concur with the tive CNS diseases like AD, characterized by b-amyloid observation that VEGF mRNA was overexpressed in deposits, neuronal death is accompanied by proliferation of retinal astrocytes of diabetic rats [46].

astrocytes, which react in an attempt to detoxify neuronal Although our study focuses on increased expression of debris, toxins and produce various trophic substances VEGF in reactive astrocytes, it is evident that there are [33,63]. Furthermore, decreased cerebral perfusion and several cell types capable of producing VEGF, that are also glucose metabolism found in AD may also induce the prevalent in reactive astrocytosis. These include, platelets, reactive astrocytes to up-regulate VEGF in an attempt to neurons, macrophages / microglia, smooth muscles around

increase neoangiogenesis (Fig. 1) [33,63]. vessel walls, megakaryocytes and polymorphonuclear

The overall results of the mouse–needle–stick injury leukocytes [36,44,46,52,54,66,72]. There is some con-model are supportive of a temporal correlation between troversy as to which of these cell types are the pre-reactive astrocytosis, neoangiogenesis and VEGF expres- dominant expressors of VEGF following damage to the sion. All three reached maximal levels between days 5–7, CNS. Plate et al. identified cells of the microglial / macro-with subsequent return towards basal levels by day 9 (Fig. phage lineage to be the major source of VEGF upregula-3). What is not obvious is why the MVD peaked first (day tion following a middle cerebral artery occlusion (MCAO) 5), followed by VEGF (day 6) and then GFAP-positive and reported virtually no VEGF production in neurons or reactive astrocytes (day 7) (Fig. 3). Perhaps the initial reactive astrocytes [54]. Lennmyr et al. demonstrated the wave of neoangiogenesis on day 5 reflects the response to most striking changes in VEGF production following inflammatory cells induced by the injury in these immuno- MCAO to be in neurons, with only weak-to-moderate competent CD1 mice, with release of other angiogenic VEGF immunoreactivity in monocytes and astrocytes [36]. stimulants in addition to VEGF [52]. The GFAP:GFP The reasons for these differences are unclear but may be transgenic mice subjected to a similar needle stick injury, attributed to differences in methods and use of reagents. clearly demonstrated co-labeling of VEGF to the GFAP Interestingly however, using the same reagents and

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con-Fig. 4. Co-localization of VEGF in reactive astrocytes of GFAP:GFP transgenic mice. Photomicrograph (scale bar depicts 20mm in length) demonstrating the co-localization of reactive astrocytes and VEGF expression in GFAP:GFP transgenic mouse brains post-stereotactic needle stick injury. (*) denotes the area of the tract injury. (A) Endogenous reactive astrocytes (arrows) emit intense green fluorescence. Typical astrocyte morphology with cell bodies and numerous processes can be seen. (B) Double labeling on the same section with anti-GFAP antibody and endogenous GFP immunofluorescence. The reactive astrocytes are stained yellow (arrows) due to the combination of the GFP green fluorescence and the anti-GFAP antibody detected via a Cy3-conjugated secondary antibody. (C) Double labeling on an adjacent section showing the co-localization of GFP-positive reactive astrocytes and VEGF protein expression as demonstrated by the red immunofluorescence staining (arrows). (D) The non-injured contra-lateral hemisphere of the brain in the same area demonstrated no significant GFP-positive green astrocytes.

ditions, we were able to detect increased VEGF immuno- angiogenic phenotype is activated when the normal bal-reactivity but unable to co-localize VEGF to reactive ance that exists between inducers and inhibitors of angio-astroctyes in GFAP:GFP transgenic and CD1 mice with a genesis is disrupted [4,21,51]. This induction results in MCAO (data not shown). This data suggests that there may angiogenesis through chemotactic and mitogenic effects on be differences in VEGF immunolocalization in the stab endothelial cells which line blood vessels [21]. A number injury and focal ischemia models [46]. Further evidence of peptides are known to be angiogenic in vivo [23]. indicative of differences in VEGF immunolocalization in However, VEGF, a homodimeric glycoprotein is a mitogen cerebral infarct is seen in our human data, where despite that specifically acts on endothelial cells and is postulated increased levels of VEGF immunoreactivity, the correla- to be the main positive regulator of angiogenesis in vivo tion to reactive astrocytosis was not as strong as was the [55,60,70]. There are many regulators of VEGF, with correlation in other neuropathologies including head injury hypoxia being the most potent physiological inducer of and brain abscess. However, this is only speculation and VEGF [19,20,28,38,62]. The factors responsible for in-requires further studies with the appropriate controls. creased VEGF production by astrocytes after damage to Angiogenesis is the sprouting of capillaries from pre- the CNS are unknown. However, hypoxia may be the existing blood vessels and during embryonic development common regulator of VEGF expression by reactive as-is a fundamental process in the formation of the vascular trocytes in response to a variety of CNS insults that create system. In the adult, angiogenesis plays a crucial role in a relative hypoxic gradient in the surrounding brain. normal and pathological processes such as wound healing, Whether modulation of angiogenic factors such as VEGF diabetic retinopathy and tumorigenesis [1,10,23]. The would favor repair in various non-neoplastic CNS

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The authors would like to thank Dr. A. Messing _{[16] N. Ferrara, Vascular endothelial growth factor, Eur. J. Cancer 32A} (Madison, Wisconsin) for the GFAP:GFP mice. We wish (1996) 2413–2422.

[17] N. Ferrara, K. Carver-Moore, H. Chen, M. Dowd, L. Lu, K.S.

also to thank Ms. T. Nicklee (Toronto, ON) for her

O’Shea, L. Powell-Braxton, K.J. Hillan, M.W. Moore, Heterozygous

assistance with the image analysis system. LA was

sup-embryonic lethality induced by targeted inactivation of the VEGF

ported by a National Cancer Institute of Canada and an

gene, Nature 380 (1996) 439–442.

American Association of Neurological Surgeons Research _{[19] G. Finkenzeller, A. Technau, D. Marme, Hypoxia induced} transcrip-Fellowship. BS was supported by the Lunenfeld Research tion of the vascular endothelial growth factor gene is independent of functional AP-1 transcription factor, Biochem. Biophys. Res.

Com-Institute summer studentship. AG is a Medical Research

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Council of Canada Clinician Scientist and this work was

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supported by operating grants to AG from Physician

discovery, Perspect. Bio. Med. 29 (1) (1985) 10–37.

Scientist Inc. and Heart & Stroke Fund of Ontario. _{[21] J. Folkman, Angiogenesis in cancer, vascular, rheumatoid and other}

diseases, Nature Med. 1 (1995) 27–31.

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form scars by extending their long, thick cytoplasmic

neoangiogenesis (MVD) and VEGF immunoreactivity in a

processes toward the site of the lesion [29]. It is likely that

variety of human CNS pathologies (see Table 1, Fig. 1).

the glial scar can serve to separate normal brain from the

Reactive

astrocytosis,

neoangiogenesis

and

increased

surrounding damaged tissue, but its formation may also be

VEGF was maximal in response to bacterial brain abscess

harmful by essentially creating a mechanical barrier [50]

and probably reflects the acuteness of the illness and the

which may impede neuronal dendritic growth, remodeling

type of response that it induces in the CNS. In an abscess,

and axonal regeneration [25,29,50,63,71]. In addition, the

in response to the released toxins, there is an inflammatory

glial scar may contribute to electrical instability in the

cellular response, components of which themselves release

region and in turn promote seizure activity [50]. However,

VEGF and other inflammatory cytokines modulators. This

more recent evidence suggests that astrogliosis may actual-

leads not only to a vigorous reactive astrogliotic response

ly facilitate CNS recovery through neurotrophic factor

but it is also one of the few CNS diseases that induces a

production around the area of the lesion [2,34,67]. In

collagen fibrovascular response.

summary, like the common collagen fibrovascular wound

The amount of VEGF immunoreactivity and MVD

healing process, reactive astrogliosis serves a beneficial

exceeded the degree of reactive astrocytosis in the brain

CNS reparative function, however, under certain circum-

around carcinomas (Table 1), perhaps attributable to the

stances it may be detrimental much like exuberant scar and

additive expression of VEGF from the GFAP positive

keloid formation in other tissues.

reactive astrocytes and the carcinoma cells themselves

This study demonstrates, using human specimens and

[22]. In cerebral infarcts, irrespective of whether it was

experimental models of CNS injury, that reactive as-

acute, subacute or old, VEGF immunoreactivity was higher

trocytosis is accompanied by a neoangiogenic response,

than the degree of reactive astrocytosis or neoangiogenic

similar to the collagen fibrovascular response elsewhere.

response (see Table 1). This may reflect the potent hypoxic

Furthermore, we speculate based on our results that a

induction of VEGF expression by the surviving reactive

potential source of this angiogenic response lies in in-

astrocytes [15], without a concomitant increase in the

creased VEGF secretion by the quiescent reactive as-

number of astrocytes or the ability to recruit new blood

trocytes. This conclusion is based on the overall consistent

vessels due to the cerebral ischemia. This data is similar to

relationship between GFAP-positive reactive astrocytes,

results in a study by Plate et al., where in a middle cerebral

(2)

artery occlusion in rats, VEGF mRNA expression was

positive reactive astrocytes. Our results confirm previous

consistently greater across different time points than the

findings that reactive astrocytes after a stab wound injury

number of CD31 stained vessels [54]. In neurodegenera-

are VEGF positive [46,52] and also concur with the

tive CNS diseases like AD, characterized by

b

-amyloid

observation that VEGF mRNA was overexpressed in

deposits, neuronal death is accompanied by proliferation of

retinal astrocytes of diabetic rats [46].

astrocytes, which react in an attempt to detoxify neuronal

Although our study focuses on increased expression of

debris, toxins and produce various trophic substances

VEGF in reactive astrocytes, it is evident that there are

[33,63]. Furthermore, decreased cerebral perfusion and

several cell types capable of producing VEGF, that are also

glucose metabolism found in AD may also induce the

prevalent in reactive astrocytosis. These include, platelets,

reactive astrocytes to up-regulate VEGF in an attempt to

neurons, macrophages / microglia, smooth muscles around

increase neoangiogenesis (Fig. 1) [33,63].

vessel walls, megakaryocytes and polymorphonuclear

The overall results of the mouse–needle–stick injury

leukocytes [36,44,46,52,54,66,72]. There is some

con-model are supportive of a temporal correlation between

troversy as to which of these cell types are the

pre-reactive astrocytosis, neoangiogenesis and VEGF expres-

dominant expressors of VEGF following damage to the

sion. All three reached maximal levels between days 5–7,

CNS. Plate et al. identified cells of the microglial /

macro-with subsequent return towards basal levels by day 9 (Fig.

phage lineage to be the major source of VEGF

upregula-3). What is not obvious is why the MVD peaked first (day

tion following a middle cerebral artery occlusion (MCAO)

5), followed by VEGF (day 6) and then GFAP-positive

and reported virtually no VEGF production in neurons or

reactive astrocytes (day 7) (Fig. 3). Perhaps the initial

reactive astrocytes [54]. Lennmyr et al. demonstrated the

wave of neoangiogenesis on day 5 reflects the response to

most striking changes in VEGF production following

inflammatory cells induced by the injury in these immuno-

MCAO to be in neurons, with only weak-to-moderate

competent CD1 mice, with release of other angiogenic

VEGF immunoreactivity in monocytes and astrocytes [36].

stimulants in addition to VEGF [52]. The GFAP:GFP

The reasons for these differences are unclear but may be

transgenic mice subjected to a similar needle stick injury,

attributed to differences in methods and use of reagents.

clearly demonstrated co-labeling of VEGF to the GFAP

Interestingly however, using the same reagents and

(3)

ditions, we were able to detect increased VEGF immuno-

angiogenic phenotype is activated when the normal

bal-reactivity but unable to co-localize VEGF to reactive

ance that exists between inducers and inhibitors of

angio-astroctyes in GFAP:GFP transgenic and CD1 mice with a

genesis is disrupted [4,21,51]. This induction results in

MCAO (data not shown). This data suggests that there may

angiogenesis through chemotactic and mitogenic effects on

be differences in VEGF immunolocalization in the stab

endothelial cells which line blood vessels [21]. A number

injury and focal ischemia models [46]. Further evidence

of peptides are known to be angiogenic in vivo [23].

indicative of differences in VEGF immunolocalization in

However, VEGF, a homodimeric glycoprotein is a mitogen

cerebral infarct is seen in our human data, where despite

that specifically acts on endothelial cells and is postulated

increased levels of VEGF immunoreactivity, the correla-

to be the main positive regulator of angiogenesis in vivo

tion to reactive astrocytosis was not as strong as was the

[55,60,70]. There are many regulators of VEGF, with

correlation in other neuropathologies including head injury

hypoxia being the most potent physiological inducer of

and brain abscess. However, this is only speculation and

VEGF [19,20,28,38,62]. The factors responsible for

in-requires further studies with the appropriate controls.

creased VEGF production by astrocytes after damage to

Angiogenesis is the sprouting of capillaries from pre-

the CNS are unknown. However, hypoxia may be the

existing blood vessels and during embryonic development

common regulator of VEGF expression by reactive

as-is a fundamental process in the formation of the vascular

trocytes in response to a variety of CNS insults that create

system. In the adult, angiogenesis plays a crucial role in

a relative hypoxic gradient in the surrounding brain.

normal and pathological processes such as wound healing,

Whether modulation of angiogenic factors such as VEGF

diabetic retinopathy and tumorigenesis [1,10,23]. The

would favor repair in various non-neoplastic CNS

(4)

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VEGF. Therefore, the functional importance of reactive

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panying reactive gliosis requires further experimentation.

1008.

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Acknowledgements

(VEGF) by astrocytoma cells is mediated by ras, Int. J. Cancer 81 (1999) 118–124.

The authors would like to thank Dr. A. Messing

_{[16] N. Ferrara, Vascular endothelial growth factor, Eur. J. Cancer 32A}

(Madison, Wisconsin) for the GFAP:GFP mice. We wish

(1996) 2413–2422.

[17] N. Ferrara, K. Carver-Moore, H. Chen, M. Dowd, L. Lu, K.S.

also to thank Ms. T. Nicklee (Toronto, ON) for her

O’Shea, L. Powell-Braxton, K.J. Hillan, M.W. Moore, Heterozygous

assistance with the image analysis system. LA was

sup-embryonic lethality induced by targeted inactivation of the VEGF

ported by a National Cancer Institute of Canada and an

gene, Nature 380 (1996) 439–442.

American Association of Neurological Surgeons Research

_{[19] G. Finkenzeller, A. Technau, D. Marme, Hypoxia induced}

transcrip-Fellowship. BS was supported by the Lunenfeld Research

tion of the vascular endothelial growth factor gene is independent of functional AP-1 transcription factor, Biochem. Biophys. Res.

Com-Institute summer studentship. AG is a Medical Research

mun. 208 (1995) 432–439.

Council of Canada Clinician Scientist and this work was

[20] J. Folkman, Toward an understanding of angiogenesis: search and

supported by operating grants to AG from Physician

discovery, Perspect. Bio. Med. 29 (1) (1985) 10–37.

Scientist Inc. and Heart & Stroke Fund of Ontario.

_{[21] J. Folkman, Angiogenesis in cancer, vascular, rheumatoid and other} diseases, Nature Med. 1 (1995) 27–31.

[22] J. Folkman, M. Klagsbrun, Angiogenic factors, Science 235 (1987) 442–447.

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