VEGF-A Angiogenesis Induces a Stable

Journal of Neuropathology and Experimental Neurology Copyright q 2004 by the American Association of Neuropathologists

Vol. 63, No. 8 August, 2004 pp. 841 855

VEGF-A Angiogenesis Induces a Stable Neovasculature in Adult Murine Brain S. I. STIVER, MD, PHD, X. TAN, PHD, L. F. BROWN, MD, E. T. HEDLEY-WHYTE, MD,

AND

H. F. DVORAK, MD

Abstract. Angiogenesis is a critical component of stroke, head injury, cerebral vascular malformation development, and brain tumor growth. An understanding of the mechanisms of adult cerebral angiogenesis is fundamental to therapeutic vessel modulation for these diseases. To study angiogenesis in the central nervous system, we injected an adenoviral vector engineered to express vascular endothelial growth factor (VEGF-A164) into adult murine striatum. Vector-infected astrocytes expressed VEGF-A164 resulting in vascular permeability, hemorrhage, and the formation of greatly enlarged ‘‘mother’’ vessels. Subsequently, endothelial cells and pericytes lining mother vessels proliferated and assembled into glomeruloid bodies, complex cellular arrays interspersed by small vessel lumens. As VEGF-A164 expression declined, glomeruloid bodies involuted through apoptotic processes to engender numerous small daughter vessels. Characterized by modestly enlarged lumens with prominent pericyte coverage, daughter vessels were distributed with a density greater than normal cerebral vessels. Daughter vessels remained stable and patent to 16 months and represented the final stage of VEGF-A-induced cerebral angiogenesis. Together, these findings provide a mechanistic understanding of angiogenesis in cerebral disease processes. Furthermore, the long-term stability of daughter vessels in the absence of exogenous VEGF-A164 expression suggests that VEGF-A may enable therapeutic angiogenesis in brain. Adenovirus; Angiogenesis; Brain; Cerebrovascular disorders; Glioblastoma; Vascular endothelial growth factor

INTRODUCTION Insults to brain such as cerebral infarction and head trauma are accompanied by an angiogenic response as part of the pathophysiological repair process (1–8). Angiogenesis is also an integral component of glioma tumor growth, and indeed endothelial hyperproliferation serves as a diagnostic criterion for glioblastoma multiforme tumors (9, 10). In these processes, vascular endothelial growth factor (VEGF-A) acts as a key angiogenic stimulus for new blood vessel growth (4, 6, 7). VEGF-A has also been implicated in the pathogenesis of cerebral cavernous and arteriovenous vascular malformations during development (11, 12). Given the importance of angiogenesis in cerebral disease, a mechanistic understanding of vessel formation in brain is essential to enable methodologies for vessel ablation or augmentation in the treatment of these pathological processes. While the role of VEGF-A during de novo blood vessel formation has been defined in developing brain (13– 15), few studies have addressed the angiogenic process in the adult cerebrum. Because of the tissue-specific nature of the angiogenic response, the mechanisms of cerebral angiogenesis can not be simply inferred from studies in non-neural organs (16). In previous work, VEGF-A From Departments of Pathology and Surgery (SIS, XT, LFB, HFD), Beth Israel Deaconess Medical Center and Harvard Medical School and The Department of Pathology (Neuropathology) (ETH-W), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts. Correspondence to: Dr. S. Stiver, Research North, Room 289, Beth Israel Deaconess Medical Center, 99 Brookline Avenue, Boston, MA 02215. E-mail: [email protected] This work was supported by US Public Health Service Grants NS02236 (SIS), HL-64402 (HFD), and AG-05134 (ETH-W) and by the Beth Israel Hospital Pathology Foundation, Inc.

expressed by an adenoviral construct in the striata of immunocompetent Fisher rats has been shown to induce angiogenic vessels (17). However, vessel formation was attributed to an associated immune and inflammatory response, as parallel studies with minipump VEGF-A treatment did not evoke an angiogenic reaction. More recent studies have shown that both VEGF-A minipump infusion and adenoviral-VEGF-A act to induce a cytokine-mediated angiogenic reaction in brain (18–21). Together, VEGF-A was found to evoke changes in vascular permeability and an increase in vessel density. However, further investigation is necessary to more fully characterize the nature, mechanistic processes, temporal profile, and long-term stability of the angiogenic vessels formed in response to VEGF-A treatment in brain. Therefore, the goal of this study was to determine the detailed steps and mechanisms of adenoviral VEGF-Ainduced cerebral angiogenesis using an adult murine model. Furthermore, we set out to define the long-term course of the angiogenic response and specifically to test whether VEGF-A could induce the formation of a mature, stable vasculature in adult brain. Immunocompromised animals were used to obviate the T cell-mediated inflammatory response evoked by adenoviral vectors in immunocompetent animals (22). We found that AdVEGF-A164 inoculation of nude mouse striatum evoked changes in the nascent vasculature resulting in the sequential formation of mother vessels, glomeruloid bodies, and daughter vessels. Daughter vessels persisted for the length of our study (16 months), long after Ad-VEGFA164 expression had subsided. The vascular structures we observed establish a model for understanding angiogenesis associated with glioma brain tumors, vascular malformation development, and reactive angiogenesis to injury. Furthermore, the persistence of daughter vessels in

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Key Words: (VEGF).

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the absence of ongoing exogenous VEGF-A stimulation provides a foundation for therapeutic vessel augmentation within the adult cerebral vasculature. MATERIALS AND METHODS Adenoviral Vectors and Animal Inoculation

Tissue Processing and Immunohistochemistry Mice were anesthetized and brain specimens fixed in situ or by standard fixation-perfusion techniques using 4% paraformaldehyde or a mixture of 2% paraformaldehyde/2.5% glutaraldehyde. Giemsa-stained Epon sections of 1.0-mm thickness were prepared as previously described (24). Collagen was stained using a Masson Trichrome Accustain kit (Sigma, St. Louis, MO). Alcian blue and Alcian blue-periodic acid-Schiff (PAS) stains followed standard protocols. b-galactosidase activity in control lacZ specimens was detected on frozen sections incubated with a solution of X-gal (1 mg/mL), 10% potassium ferricyanide, and 10% potassium ferrocyanide (pH 8.0) at room temperature for 2 hours. Vessels in X-gal-treated sections were visualized by subsequent CD-31 immunostaining. Primary antibodies against endothelial cells employed CD31 (PECAM; Pharmingen, San Diego, CA) and von Willebrand

J Neuropathol Exp Neurol, Vol 63, August, 2004

In Situ Hybridization In situ hybridization (ISH) was performed on 6-mm frozen sections with RNA probes against VEGF-A, Flt-1 (VEGFR-1), Flk-1 (VEGFR-2), angiopoietin1 (Ang1), angiopoietin2 (Ang2), Tie1, Tie2, thrombospondin1 (Tsp1) and thrombospondin2 (Tsp2) as previously described (30–35). For combined immunohistochemistry and ISH, slides were incubated first with RNA probe and washed sequentially with 2X SSC/50% formamide/10 mM dithiothreitol at 508C, 4X SSC/10 mM Tris/1 mM EDTA with 20 mg/ml ribonuclease at 378C, 2X SSC/50% formamide/10 mM dithiothreitol at 658C, and 2X SSC. Slides then were immunostained as above, dehydrated in graded alcohols containing 0.3 M ammonium acetate, dried, coated with Kodak NTB 2AR emulsion (Kodak-Lab, Wilmington, NC), incubated


Nonreplicating adenoviral serotype 5 vectors containing encoding sequences for VEGF-A164 and lacZ were constructed as previously described (16). We chose to over-express VEGF-A164 as it is the most commonly expressed murine isoform of VEGFA. The 164 isoform is also moderately diffusible (23). Adenoviral vectors (6 3 105 pfu) in 0.1 M Tris hydrochloride (pH 8.0) were injected stereotactically into the striata of 4- to 6week-old female athymic Nu/Nu mice (National Cancer Institute, Frederick, MD). To minimize tissue trauma, injections were performed with Hamilton syringes fitted with custommade 30 gauge needles (Hamilton Company, Reno, NV). Stereotactic injections were made 2.3 mm lateral, 0.5 mm anterior, and 2.4 mm deep, relative to the bregma. Specimens were examined in triplicate for each study parameter at time points: 12, 24, and 36 hours, 2, 3, 4, 5, 8, 10, 14, 17, 21, 28, 37 days, and 3, 6, 10, and 16 months. Control mice were similarly injected with comparable amounts of adenoviral vectors expressing lacZ, and specimens were analyzed in triplicate at time points 12 and 24 hours, 2, 3, 5, 10, 14, 21, 37 days, and 3, 6, and 16 months. In our initial experiments, DiI (1,19-dilinoleyl-3,3,39,39-tetramethylindocarbocyanine 4-chlorobenzenesulfonate salt; Molecular Probes, Eugene, OR) added to phosphate buffered saline (PBS) vehicle confirmed the accuracy of our stereotactic coordinates. To determine whether the needle trauma itself could induce a significant angiogenic reaction, we introduced our 30 gauge needles into mouse striatum and using the vernier adjustments on the stereotactic frame, performed small repeated movements of the needle. The needle was removed, displaced fractions of a mm horizontally, reintroduced, and similarly manipulated 2 or 3 additional times. Needle injury experiments were performed in triplicate and specimens were examined as 1-mm Epon sections and by CD31 immunohistochemistry at days 5 and 28 and at 10 months. Animal protocols were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee.

factor (Santa Cruz Biotechnology, Santa Cruz, CA). Basement membrane antibodies to laminin and collagen IV were purchased from Chemicon International (Temecula, CA). As no single specific marker exists to detect pericytes, we employed a panel of antibodies comprising chondroitin sulphate proteoglycan (NG2; Chemicon International), Griffonia simplicifolia lectin I (Vector Laboratories, Burlingame CA), and platelet-derived growth factor (PDGF)-b receptor (Santa Cruz Biotechnology) (25–27). a-Smooth muscle actin antibodies were purchased from Sigma. Antibodies to 29,39-cyclic nucleotide 39-phosphodiesterase (CNPase; Chemicon International) and glial fibrillary astrocytic protein (GFAP; Zymed, South San Francisco, CA) were used to specifically detect oligodendrocytes and astrocytes, respectively. The macrophage antibodies, CD 11b and F4/80, were purchased from Serotec (Raleigh, NC). Antibodies to fibrinogen (Chemicon International) recognize both fibrinogen and fibrin; therefore, to stain selectively for fibrin, we extracted fibrinogen and soluble clotting products with 4% formalin/2% acetic acid, a process that retained insoluble, cross-linked fibrin (28). Antibodies specific for the cleaved form of caspase-3 (Cell Signaling Technology, Beverly, MA) were employed in our apoptosis studies (29). TUNEL staining was performed using an Apoalert DNA fragmentation kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. Immunohistochemical studies were performed on 6-mm frozen sections. Following treatment with blocking serum for 1 hour, sections were incubated with primary antibody in PBS at 48C overnight. Optimal antibody concentrations were determined by serial dilution. Sections were treated with biotinylated secondary antibody for 30 min, and the reaction product was visualized by avidin-biotin complex (Vectastain ABC elite kit; Vector Laboratories) and 3,39-diaminobenzidine tetrahydrochloride (DAB) against a light Mayer’s hematoxylin counterstain. Immunofluorescence studies employed Texas red and FITC (Vector Laboratories), together with Alexa Fluor 488, 568, and 594 (Molecular Probes), secondary antibodies. Fluorescence images obtained from Zeiss Axiophot (Carl Zeiss Microscopy, Germany) and Nikon TE300 (Nikon, Melville, NY) microscopes were processed using IPLab software (Scanalytics, Fairfax, VA). Gross and photomicrograph images were captured with a digital imaging system and Spot software (Diagnostic Instruments, Sterling Heights, MI) and processed with Adobe Photoshop 7.0.

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in the dark for 4 weeks at 48C, developed, and counterstained with Mayer’s hematoxylin.

Quantitative RT-PCR and Protein Expression Analyses

Permeability, Perfusion, and Cell Proliferation Studies To assess vascular permeability, 2% Evans blue dye in saline (0.2 mL) was injected intravenously (i.v.) into animals at time points to 21 days. Brain specimens, harvested 30 min later,

Statistical Analyses Differences in RT-PCR relative Ct values and ELISA VEGFA protein concentrations were assessed by t-test analysis. Mann Whitney Rank Sum analysis with Dunn’s method of multiple comparison was used to evaluate differences in quantitative vessel measurements. Analyses were performed with a SigmaStat software program (SPSS, Chicago, IL) and significance considered at p , 0.05.

RESULTS VEGF-A Expression in Adenoviral-Infected Astrocytes Within 24 hours of inoculation, Ad-VEGF-A164-infected cells in the striatum and corpus callosum began to express VEGF-A mRNA (Table 1). By in situ hybridization, strong expression was evident from days 3 to 14, and thereafter expression progressively declined with low mRNA levels detected at day 35 (Fig. 1; Table 1). At 3, 6, and 16 months we found no ISH evidence of exogenous VEGF-A mRNA expression. Similarly, in Ad-lacZtreated specimens, reaction with X-gal demonstrated strong lacZ expression at days 2 through 21, with significant tapering in sections at day 35. Transgene staining was not found in striata of Ad-lacZ specimens at 16 months. Quantitative RT-PCR analysis of VEGF-A mRNA levels confirmed intense adenoviral VEGF-A expression during the first week, with peak increases greater than J Neuropathol Exp Neurol, Vol 63, August, 2004


To determine the temporal profile of VEGF-A mRNA expression in our model, we performed quantitative RT-PCR studies. Mice were euthanized by cervical dislocation without anesthesia and coronal sections (2 mm) were quickly harvested using a brain matrix. A 4-mm biopsy punch was then used to obtain reproducibly sized specimens (;25 mg) centered on the reaction site. Specimens were homogenized and total RNA isolated using a RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was prepared by reverse transcription with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). Primers for murine VEGF-A and the mouse housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Primer3, a primer picking software program designed by the Whitehead Institute (Cambridge, MA) and Howard Hughes Medical Institute, available at www.frodo.wi.mit.edu/primer3. Primers crossed intron–exon boundaries to reduce amplification of genomic DNA. The sequences for the forward and reverse primers were synthesized by Integrated DNA Technologies (Coralville, IA) as follows: mouse VEGF-A (forward 59-GTA CCT CCA CCA TGC CAA GT-39; reverse 59ACT CCA GGG CTT CAT CGT TA-39) and mouse GAPDH (forward 59-AAG GTC ATC CCA GAG CTG AA-39; reverse 59 AAG TCG CAG GAG ACA ACC TG-39). Quantitative RT-PCR experiments employed Sybr-green detection (Applied Biosystems, Foster City CA) with thermal cycling conditions of 508C 3 2 min, denaturation at 958C 3 10 min, and 40 cycles at 958C for 15 s alternating with 608C for 1 min performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems). Ct values were determined in the linear exponential phase of amplification and the comparative Ct method was used to determine the relative quantities of mRNA. The relative fold changes in transcript were reported in comparison to normal brain, whose fold change value was set to 1. Results were obtained from 3 specimens per time point and each specimen was run in duplicate. Specimens for VEGF-A enzyme-linked immunosorbent assays (ELISA) obtained from fresh coronal sections of 2 mm thickness and 4 mm biopsy excision of the reaction site were homogenized in 50 mM Tris (200 mL) containing protease inhibitor cocktail (10 mL/mL) (Sigma). Supernatant solutions (50 mL) rendered following centrifugation at 14,000 g 3 30 min at 48C were analyzed for VEGF-A with a Quantikine Mouse VEGF immunoassay kit (R&D Systems, Minneapolis, MN). As described in the manufacturer’s protocol, optical density was measured at 450 and 570 nm to quantitate VEGF-A concentrations spectrophotometrically from a standard curve. VEGF-A measurements were normalized to total protein concentration (Bio-Rad, Hercules, CA). Triplicate samples were analyzed for each time point and each specimen was run in duplicate.

were sectioned in 2-mm coronal slices and photographed. Frozen sections were mounted in fluoromount (Southern Biotechnology Associates, Birmingham, AL) and examined by fluorescence microscopy. For perfusion studies, colloidal carbon and FITC-Lycopersicon esculentum lectin (Vector Laboratories) were injected as tracers into animals. A suspension of 0.25% carbon in saline (0.2 mL) was injected i.v. and allowed to circulate for 30 to 45 min, following which animals were euthanized and tissues prepared for 1-mm Epon sections. FITC-Lycopersicon esculentum lectin (50% in saline, 0.2 mL) was circulated for 30 min following tail vein injection, and frozen sections examined by immunofluorescence. For quantitative measurements, images of the reactive site in lectin-perfused specimens were taken at 340 magnification over 10 contiguous fields measuring 0.65 mm2. For each of the Ad-VEGF-A, AdlacZ, and normal striata, at least 240 images were obtained from 6 sections from each of 4 different animals. Images were analyzed using IPLab image analysis software (Scanalytics). Vessel diameters were calculated as the minor axis of ellipses conformed to nonbranching vessel segments. To further verify these data, direct measurements of vessel diameters were performed midway along 5 representative vessels in longitudinal profile in each of the images analyzed. To identify proliferating cells, 25 mCi 3H-thymidine (Perkin Elmer Life Sciences, Boston, MA) in 0.25 mL of saline was injected i.v. Animals were harvested 1 hour later for preparation of 1-mm Epon sections that were coated with Kodak NTB 2AR emulsion (Kodak-Lab), incubated for 4 weeks in the dark, and lightly counterstained with Giemsa.

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TABLE 1 In Situ Hybridization Results for Ad-VEGF-A164-Induced Angiogenesis in Striatum* Time

Predominant Vascular Structure†

Day 12 h 24 h 2 Mother Vessels 3 5 Glomeruloid Bodies 8 10 14 Daughter Vessels 21 28 37 3 mo 16 mo

VEGF-A 1 11 11 111 111 111 111 111 11 11 1

Flk-1

Flt-1

1 11 111 111 111 111 11 11 11 1 n/a n/a

1 11 111 111 111 111 1 11 11 1 n/a n/a

Ang1

1 6 1 6 n/a n/a

Ang2

111 111 111 11 11

Tie1

1 11 11 11 11

1 n/a n/a

n/a n/a

Tie2

1 11 11 11 11 1 1 6 n/a n/a

Tsp1

6 1 11 111 111 11 1

n/a n/a

Tsp2

1 1 1 6

n/a n/a

100-fold at day 3 (Table 2). Between days 10 and 21, relative expression levels were 2- to 3-fold that of normal brain, but these differences were not statistically significant. VEGF-A protein expression determined by ELISA assay exhibited a similar trend (Table 3). Peak levels at days 3 to 5 were significantly elevated as compared to Ad-lacZ-treated and normal striatum. Levels then quickly declined, and mildly elevated expression at day 21 was not statistically different from normal striatum. The temporal profile of VEGF-A mRNA and protein expression that we observed is consistent with previous studies that have demonstrated intense transgene expression for the first week, followed by progressive decline leading to weak expression at 6 to 8 weeks following intracerebral adenoviral inoculation (36–38). Combined in situ hybridization and GFAP immunohistochemistry demonstrated that the majority of VEGFA mRNA-producing cells were astrocytes. A portion of the VEGF-A mRNA-producing cells did not colocalize with GFAP immunosignal and suggested adenoviral infection of a small cohort of neurons or microglial (36, 39, 40). VEGF-A mRNA expression was accompanied by robust expression of the VEGF-A receptors, Flk-1 and Flt-1 (Fig. 1; Table 1). Traumatic brain injury induced by large needle, freeze, and weight-drop injury has been demonstrated to upregulate VEGF-A expression and induce an angiogenic response primarily at the cortical surface (4, 5, 41, 42). In our study, the smaller 30 gauge needles employed evoked a transient and mild upregulation of VEGF-A. This response was weak and insignificant in comparison to the robust VEGF-A expression we observed throughout the J Neuropathol Exp Neurol, Vol 63, August, 2004

striatum following Ad-VEGF-A164 treatment. In AdVEGF-A164-treated specimens VEGF-A mRNA extended throughout the striatum. In contrast, VEGF-A mRNA in control animals was observed only in scattered cells immediately adjacent to the needle tract and was barely detected above background levels. Furthermore, Flt-1 and Flk-1 mRNAs were not detected in our Ad-lacZ specimens, nor did we identify any expression of Ang1 or 2, Tie1 or 2, or Tsp1 or 2 mRNAs. Mildly increased peak VEGF-A levels determined by ELISA assay in Ad-lacZtreated striatum were not statistically different from those measured in normal striatum (Table 3). Vascular Permeability, Hemorrhage, and Immune Response Ad-VEGF-A164 induced a brisk angiogenic response in striatum at a 2-log lower viral dose than that typically used in ear skin (16) (Fig. 2A–E). Vascular permeability, edema, and hemorrhage accompanied VEGF-A164 expression. Intravenously administered Evans blue dye extravasated extensively into brain parenchyma at days 3 to 5 and continued at lower levels to day 14, indicating significant blood vessel hyperpermeability (Fig. 2F). Fibrin, detected immunohistochemically, was deposited in the neuropil and evidenced the extravasation and clotting of plasma fibrinogen from hyperpermeable vessels (data not shown). As a consequence of vascular hyperpermeability, edema formation led to mild swelling of the ipsilateral hemisphere. In addition, numerous red blood cells infiltrated the adjacent neuropil at days 2 to 5, and indeed, coronal sections at day 5 appeared hemorrhagic to the


* Grading scale: 111, strong signal at multiple sites; 11, easily detected at multiple sites; 1, distinguishable above background; 6, present in some but not all specimens; blank, undetectable; n/a, not tested. Data are representative of specimens from 4 animals at each time point. † Detected mRNA signal is not necessarily associated with the predominant vascular structure noted. See Results for further details.

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TABLE 2 Quantitative RT-PCR Analyses of Ad-VEGF-A164-Treated Striatum Relative DCt Value Ad-VEGF-A164 versus Normal Striatum Day 3 5 10 14 21 3 mo 16 mo Normal striatum

132 53 3.1 2.4 1.7 0.5 0.7 1.0

SE

p value

22.7 15.9 1.0 0.7 0.4 0.1 0.2

,0.001 ,0.001 0.08 0.08 0.17 0.01 0.04

Ad-VEGF-A164

Fig. 1. In situ hybridization studies with 35S-labeled antisense probes 3 days following Ad-VEGF-A164 injection into striatum. Paired bright and dark field microscopy evidenced expression of VEGF-A mRNA in the parenchyma of the striatum (A); Flk-1 mRNA (B); Flt-1 mRNA (C); and Ang 2 mRNA (D) in enlarged mother vessels. Scale bars: 50 mm.

naked eye (Fig. 2B). At later times, hemosiderin, a marker of degraded extravasated hemoglobin, persisted and facilitated localization of the reactive site in long-term specimens. Together, morbidity from edema and hemorrhage at higher doses of Ad-VEGF-A164 precluded dose response studies. At lower doses it was difficult to localize an angiogenic response which, in the few specimens where it was identified, appeared qualitatively similar to the response at higher doses. We also observed a mild cellular immune reaction associated with adenoviral inoculation. Immunostaining at days 2 to 14 with the macrophage antibodies F4/80 and CD11b evidenced scattered macrophages/microglia in Ad-VEGF-A164-treated as well as control Ad-lacZ specimens, as has been previously observed by others (37)

Day

VEGF-A (pg/mg protein)

SE

3 5 10 14 21 16 mo

587.5 506.3 19.7 16.5 7.0 9.6

68 70 2.8 4.4 1.7 0.4

Ad-lacZ VEGF-A (pg/mg protein)

SE

6.7† 1.4 7.7† 0.9 2.4 ,1.0 n/a n/a Normal Striatum 4.4 1.2

p value* ,0.001 0.016 0.018 0.04 0.28 0.07

* p value for statistical comparison of Ad-VEGF-A164 versus Ad-lacZ; comparison was made to normal striatum for day 14 and later time points. † Peak VEGF-A concentrations in Ad-lacZ-treated specimens were not statistically different from those in normal striatum (p 5 0.08). n/a, not tested.

(data not shown). Some of the macrophages within AdVEGF-A164 specimens contained phagocytosed red blood cells and suggested that a portion this response was elicited by red cell extravasation. In addition, very occasional polymorphonuclear cells were found within the lumens of sporadic vessels in Ad-VEGF-A164 specimens at day 5 but not at other time points. Vessel Enlargement, Mother Vessel Formation, and Activated Endothelial Cells (Days 2 to 5) The initial morphological changes in the vasculature following Ad-VEGF-A164 injection involved nascent microvessel enlargement and the formation of thin-walled ‘‘mother’’ vessels. Vessel enlargement first appeared 48 to 72 hours after injection (Fig. 3A, B). Vessels progressively increased in diameter over the next 2 to 3 days to J Neuropathol Exp Neurol, Vol 63, August, 2004


TABLE 3 VEGF-A Protein Expression by ELISA Immunoassay in Ad-VEGF-A164 and Ad-lacZ-Treated Striatum

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yield mother vessels with diameters many times that of normal (Fig. 3C, D). Ipsilateral draining veins, remote from the area of VEGF-A expression, became enlarged possibly due to increased blood flow (Fig. 2C). By ISH, mother vessels strongly expressed typical markers of angiogenesis. Flk-1 and Flt-1, as well as Ang2 mRNAs were observed at day 3, coincident with early mother vessel enlargement (Fig. 1B–D; Table 1). Tie1 and Tie2 mRNA expression was also first detectable on day 3, but strong expression did not appear until day 5. In addition, strong Tsp1-mRNA also localized to mother vessels between days 3 and 10 (Table 1). Initially, the endothelial cells lining mother vessels were thinned, but they subsequently became activated and projected large, rounded nuclei into the enlarged vascular lumens (Fig. 3E). Endothelial cells and pericytes lining mother vessels exhibited frequent mitotic figures and labeled with tritiated thymidine as indicative of active cellular proliferation (Fig. 3F, G). As expected, enlarged vessels demonstrated typical markers of endothelium, staining strongly with antibodies to CD-31 and von J Neuropathol Exp Neurol, Vol 63, August, 2004

Willebrand factor (Fig. 3C). A focal, patchy loss of laminin- and collagen IV-staining in Ad-VEGF-A164-induced mother vessels was observed at days 3 to 4 and suggested a transient disruption of the basement membrane in enlarging vessels (Fig. 3H, I). Thereafter, staining for these basement membrane proteins was strong and uniform. In contrast, CD-31 staining of Ad-lacZ control specimens evidenced no enlargement of vessels or changes in endothelial morphology (Fig. 3D). Similarly, sections of striatum subjected to repeat fine-needle trauma evidenced normal vessels by CD-31 immunostaining. Bridged and Cavernous Vascular Structures (Days 5 to 10) The structure of mother vessels continued to evolve over the next several days. Activated endothelial cells lining some mother vessels extended cytoplasmic protrusions into and across vascular lumens to form bridged vascular structures (Fig. 3J, K). However, endothelial cell protrusions directed abluminal into the neural parenchyma, as characteristic of sprouting angiogenesis during


Fig. 2. Whole brain (C) and coronal specimens (A, B, D–F) following Ad-VEGF-A164 inoculation of striatum. A: Sections through striatum at day 3 exhibited prominent enlarged vessels. B: At day 5, the angiogenic site appeared markedly erythematous. C: Enlarged temporal veins drained the angiogenic response. In this specimen at day 5, the angiogenic site lay deep to the proximal aspect of the draining vein (arrow). Enlarged surface vessels were not observed in Ad-lacZ-treated animals. D: Vessels continued to be observed within the evolving angiogenic reaction at day 17. E: Veins draining the angiogenic site were still enlarged at day 37, and suggested that flow through the vasculature was increased. F: At day 5, intravenously administered 2% Evans Blue dye extravasated into brain parenchyma adjacent to the angiogenic response. Fresh, nonperfused specimens. Scale bars: A, C 5 1 mm; B, D, F 5 500 mm; E 5 250 mm.


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embryological cerebral vascular development, were not found. Massively enlarged vascular structures, termed ‘‘caverns,’’ also formed during this time (Fig. 3L). Caverns were many times larger than mother vessels and the presence of intravenously injected colloidal carbon within their lumens demonstrated that they were perfused entities (data not shown). The endothelium lining caverns was thin, in contrast to the activated endothelium of mother vessels at this time. Bridged and cavernous vascular structures were seldom observed as discrete structures after day 10 as they were enveloped by the ensuing proliferative glomeruloid response described below. Glomeruloid Body Formation (Days 8 to 21) Mother vessels thence evolved into glomeruloid bodies by a complex process of cell proliferation and reorganization. Glomeruloid bodies began as small collections in

the endothelial lining of mother vessels (Fig. 4A, B). The cellular mass subsequently enlarged with progressive expansion into the mother vessel lumen (Fig. 4C). Large vessel lumens were transformed as the hypercellular mass filled the mother vessel and thereby entrapped small vascular lumens (Fig. 4D). Glomeruloid bodies, comprising small capillary lumens amidst hypercellular clusters, resulted from this proliferative process. The small capillaries within glomeruloid bodies remained patent and connected to the host vasculature as evidenced by the presence of intravenous tracers, colloidal carbon and FITC-Lycopersicon esculentum lectin, within their lumens (data not shown). The incorporation of tritiated thymidine into developing and mature glomeruloid bodies evidenced active cell division (Fig. 4E). Numerous mother vessels became filled with proliferative cellular masses, and the reactive site at days 10 to 14 appeared as a field studded by glomeruloid clusters (Fig. 4F). J Neuropathol Exp Neurol, Vol 63, August, 2004


Fig. 3. VEGF-A-induced mother vessel formation, bridging, and cavernous formation (days 2–10). A: Nascent vessels in AdVEGF-A164-treated striatum were observed to enlarge beginning at day 2. B: Shown at the same magnification, vessels in the contralateral striatum (arrows) were significantly smaller at this time. C: VEGF-A induced further progressive enlargement of vessels and resultant mother vessels were demonstrated by immunohistochemistry with CD-31 antibodies. D: In contrast, control Ad-lacZ-treated specimens at day 5, assayed for b-galactosidase (blue staining) and immunostained with CD-31, demonstrated morphologically normal vessels. E: Activated endothelial cells projected enlarged nuclei into the lumens of mother vessels. F: Endothelial cells within enlarged vessels labeled with tritiated thymidine (arrow), as shown here at day 8. G: Mitotic figures were also frequently observed in enlarged vessels. H, I: Staining for laminin at day 4 (H) and collagen IV at day 3 (I) demonstrated a focal loss of basement membrane components (arrows). J: Bridges formed as activated endothelial cells extended across to other points of the vessel circumference. K: Bridging led to compartmentalization of mother vessel lumens. L: Cavernous vascular structures also formed and exhibited vessel lumens many times the diameter of mother vessels. Images of Giemsastained 1-mm sections (A, B, E–G and J–L). Specimen preparation: fixed in situ (A, B, F); fixation-perfused (C–E, G–L). Scale bars: A, B, F, G, J, K 5 10 mm; C–E, H, I, L 5 25 mm.

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Ad-lacZ specimens at these times were normal; they showed no evidence of a proliferative response and completely lacked glomeruloid structures. The cellular constituents of glomeruloid bodies comprised hyperplastic endothelial cells and pericytes. Within glomeruloid bodies, endothelial cells demonstrated immunoreactivity against CD-31 and von Willebrand factor, and pericytes were identified with antibodies to NG2, Griffonia simplicifolia lectin I, and PDGF-b receptor (Fig. 4G, H) (25–27). Because NG2 is also expressed by oligodendrocyte precursors, we confirmed that glomeruloid bodies were devoid of oligodendrocyte (CNPase) and astrocyte (GFAP) immunoreactivity (43). Masson trichrome histology determined that collagen was deposited in the extracellular matrix surrounding glomeruloid bodies (Fig. 4I). Focal, intense VEGF-A mRNA expression localized to the epicenter of glomeruloid bodies, and suggested that J Neuropathol Exp Neurol, Vol 63, August, 2004

VEGF-A was important for glomeruloid transformation (Fig. 5A). Glomeruloid body cells strongly expressed Flk-1 and Flt-1 mRNAs, as well as those of Ang2, Tie1, and Tie 2 (Fig. 5B–F). Evolution of Glomeruloid Bodies into Daughter Neovessels (Days 14 to 37) Glomeruloid bodies were transient structures and matured into daughter vessels (Fig. 6). This maturation process involved apoptosis as evidenced by TUNEL-positive staining, immunoreactivity against antibodies to the activated form (cleaved p17) of caspase-3, and cellular condensation patterns typical of apoptotic bodies (Fig. 6A– C). As glomeruloid bodies involuted, the small vascular lumens within them remained intact, thereby giving rise to daughter vessels (Fig. 6D–F). Evolving daughter vessels continued to express Tie2 mRNA (not shown). Newly formed daughter vessels were readily identified by


Fig. 4. Glomeruloid body formation (days 8–21). A: In the early stages of glomeruloid body formation, small collections of cells were observed lining the walls of mother vessels. B: Immunohistochemical staining with GFAP antibody further demarcated the glial-vessel interface and evidenced early glomeruloid formation within the vessel wall. C: The hypercellular collection thence expanded into the mother vessel lumen. D: As the cellular mass grew and expanded to fill the mother vessel, multiple small vascular lumens (arrows) became entrapped in the developing glomeruloid body. E: 3H thymidine labeled numerous cells within the constitutive cellular mass and evidenced active cellular division within developing and formed glomeruloid bodies. Small vessels were observed, juxtaposed to pericytes (arrow), within glomeruloid bodies. F: Cellular proliferative processes occurred in most mother vessels and a field studded by clusters of glomeruloid bodies resulted. G, H: The cellular constituents of glomeruloid bodies comprised endothelial cells and pericytes as demonstrated by immunofluorescence studies showing reactivity with the endothelial marker CD-31 (G, red) and the pericyte marker Griffonia simplicifolia lectin I (H, green). Glomeruloid bodies also showed immunoreactivity with antibodies against the pericyte marker, chondroitin sulphate proteoglycan (NG2) and PDGF-b receptor. I: As demonstrated by Masson trichrome staining, collagen (blue staining) was deposited in the extracellular matrix adjacent to glomeruloid bodies. Giemsa-stained 1-mm Epon sections (A, C, D–F). Specimen preparation: fixation-perfused (A, B, G–I); fixed in situ (C–F). Scale bars: A, C–E 5 10 mm; B, G, I 5 25 mm; F, H 5 50 mm.


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Fig. 5. Paired bright and dark field images of in situ hybridization studies of glomeruloid bodies (days 10–14). A: VEGF-A mRNA expression localized to the epicenter of glomeruloid bodies. B–F: Flk-1, Flt-1, Ang2, Tie1, and Tie2 mRNApositive cells were also evidenced within glomeruloid bodies. Scale bars: A, C, F 5 50 mm; B, D, E 5 25 mm.

We examined specimens at late time points to determine whether daughter vessels persisted as stable vascular entities. At 6 months and later, glomeruloid bodies were completely replaced by a daughter neovasculature comprised of numerous small prominent vessels (Fig. 7A, B). Occasional vessels with a cellular coat and enlarged many times the diameter of small daughter vessels may have represented persistent stabilized mother vessels (Fig. 7C). Daughter vessels remained stable and microscopically were unchanged at 10 and 16 months. Pericytes, evidenced in 1-mm Epon sections and by strong immunoreactivity with antibodies to NG2, richly invested daughter vessels (Fig. 7D). Daughter vessels also exhibited strong immunoreactivity with antibodies to CD-31 as well as collagen IV and laminin (Fig. 7E–H). By contrast, Ad-lacZ-treated specimens at 16 months demonstrated normal vascular morphology and density as determined by immunostaining with CD-31, collagen IV, and laminin antibodies (Fig 7F). In addition, the vasculature at the epicenter of control specimens subjected to repeat fineneedle trauma and stained with CD-31 also appeared normal without evidence of morphological change at 10 months following the traumatic insult (Fig. 7I). J Neuropathol Exp Neurol, Vol 63, August, 2004


their increased vessel density and modestly enlarged vessel lumens, as compared to normal cerebral vessels (Fig. 6F–H). By contrast, sections of Ad-lacZ-treated striatum and those of controls subjected to repeat fine-needle trauma showed no changes in vessel density or morphology at these time points (Fig. 6H). The vascular walls of AdVEGF-A164-induced daughter vessels were lined by pericytes, as demonstrated in 1-mm Epon sections and by strong perivascular immunoreactivity to NG2 antibodies (Fig. 6I). Masson trichrome staining evidenced new collagen deposition in association with evolving daughter vessels (Fig. 6J). Collagen deposits were not found in control specimens indicating that deposition of this extracellular matrix component did not represent a response to needle injury. Daughter vessels were more densely distributed than the normal cerebral vessels, and frequently they grouped in small clusters (Fig. 6E, F). Consistent with these increased vessel densities and enlarged lumen diameters, prominent ipsilateral vessels drained to the temporal lobe and inferiorly to the base of the brain (Fig. 2E). On microscopic examination we also found prominent vessels distributed about the periphery of the angiogenic reaction. Morphologically, these peripheral vessels were muscularized and exhibited thickened, multicellular walls that stained with antibodies to a-smooth muscle actin (Figs. 6K, L, 7L). Control needle injury and Ad-lacZ specimens showed no evidence of muscularized changes in the vasculature.

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An extracellular matrix distinct from normal neuropil demarcated the epicenter of the angiogenic response and facilitated localization of daughter vessels in long-term specimens (Fig. 7A). Masson trichrome stains demonstrated the presence of a significant collagenous component to this matrix (Fig. 7J). Hyaluronic acid, characteristic of the normal neuropil, was not detected in this matrix by Alcian blue and Alcian blue-PAS staining. AdlacZ and needle injury control specimens showed no evidence of collagen in the neuropil, indicating that deposition of this extracellular matrix constituent did not represent a generalized scarring response. Daughter vessels were observed to persist in the absence of ongoing, exogenous VEGF-A stimulation. In our ISH studies at 16 months, we found no evidence of VEGF-A mRNA expression above background levels. J Neuropathol Exp Neurol, Vol 63, August, 2004

Quantitative RT-PCR and ELISA analyses demonstrated that VEGF-A mRNA and protein levels at long-term endpoints were not statistically different from those of normal striatum (Tables 2, 3). Staining of daughter vessels following intravenous injection of fluorescent-labeled Lycoperiscon esculentum lectin confirmed that daughter vessels were patent and perfused even at late time points (Fig. 7K, L). About the periphery of the angiogenic site, we also continued to observe prominent vessels that were muscularized and stained strongly for a-smooth muscle actin (Fig. 7L). Daughter vessels themselves were not muscularized in appearance and did not show significant a-smooth muscle actin immunoreactivity (Fig. 7L). Daughter vessels exhibited a statistically significant increase in vessel diameter compared to normal cerebral vessels (Table 4; Fig.


Fig. 6. Evolution of glomeruloid bodies to form daughter vessels (days 14–37). A: Apoptotic bodies (arrows) were observed in association with involuting glomeruloid bodies. B: That glomeruloid body involution occurred by an apoptotic process was further evidenced by positive TUNEL-stained (green) sections of glomeruloid bodies. C: Immunohistochemistry with antibodies specific to the cleaved form of caspase-3 also stained cells (arrows) within involuting glomeruloid bodies. D: Small vessels (arrows) within involuting glomeruloid bodies remained intact during the apoptotic process, as shown here at day 21. E, F: With further glomeruloid involution, daughter vessels formed in clusters reminiscent of their glomeruloid body heritage. G: At day 28, CD-31 immunostaining evidenced an increased vascular density in Ad-VEGF-A164-treated striatum. H: In contrast, vascular density and morphology were normal in control Ad-lacZ-treated striatum as demonstrated by b-galactosidase reaction (blue) and CD-31 immunostaining. I: As shown at day 28, daughter vessels exhibited prominent pericyte coverage by NG2 immunoreactivity. J: Collagen was present in the extracellular matrix surrounding evolving daughter vessels, as evidenced by blue staining (arrows) in trichrome-processed sections. K: Large vessels with thick walls were found at the periphery of the evolving angiogenic reaction. An involuting glomeruloid body more central to the angiogenic reaction is shown in the bottom left (arrow). L: Vessels at the periphery of the angiogenic reaction appeared as multicellular structures and cross-sectional views demonstrated that these vessels have muscular walls. Giemsa-stained 1-mm Epon sections (A, D–F, K, L). Specimen preparation: fixed in situ (A, B, G, I–L); fixation-perfused specimens (C–F, H). Scale bars: A–D, I, J 5 10 mm; E, K 5 50 mm; F–H, L 5 25 mm.


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Fig. 7. Long-term sequelae of Ad-VEGF-A164-induced cerebral angiogenesis (10–16 months). A: Low-power views of striatum at 10 months and later following Ad-VEGF-A164 treatment showed changes in the extracellular matrix and numerous small vascular lumens. B: High-power view of the long-term angiogenic response demonstrated collections of small daughter vessels distributed heterogenously and with increased density as compared to normal striatum. C: Occasional enlarged vessels with a cellular coat may have represented the persistence of stabilized mother vessels. D: Daughter vessels were observed to be richly invested by pericytes. E: Clusters of daughter vessels stained with antibodies to CD-31 and evidenced an increased vessel density and vascular lumen diameter. F: In comparison, CD-31 immunochemistry of Ad-lacZ-treated striatum at 16 months demonstrated normal vessel morphology and density. G, H: Antibodies to collagen IV (G) and laminin (H) also strongly stained daughter vessels in sections of Ad- VEGF-A164-treated striatum at 16 months and further evidenced their clustered distribution and enlarged lumen diameters. I: Sections of normal striatum subjected to repeated needle injury demonstrated no changes in vascular density or morphology at 10 months following the insult. As shown here, normal striatal vessels were found at the epicenter of the injury as demarcated by hemosiderin staining (central brown staining) and a chain of pyknotic nuclei. J: Trichrome-stained sections of long-term specimens demonstrated the deposition of collagen (blue) in the extracellular matrix. K: Intravenous injection of Lycoperiscon esculentum lectin tracer (green) 16 months following Ad-VEGF-A164 inoculation demonstrated that daughter vessels remained patent and were perfused at late time points. L: a-Smooth muscle actin staining (red) of sections of Lycoperiscon esculentum lectin-perfused specimens (green) at 16 months showed that daughter vessels were not a-smooth muscle actin-positive. Large muscularized vessels, peripheral to the epicenter of the angiogenic response, did however exhibit strong a-smooth muscle actin staining (arrows). M: Box plots of vessel density measured in FITC-labeled Lycoperiscon esculentum lectin-perfused sections of Ad- VEGF-A164, Ad-lacZ, and normal striatum demonstrated a statistically significant increase in vessel density in Ad-VEGFA164-treated striatum at 16 months. Each box plot shown is representative of the vessel area as a percentage of total area (0.65 mm2) at the reactive site. For each of 4 animals, images were analyzed from 6 sections of 10 contiguous fields/section at 40 times magnification. Giemsa-stained 1-mm Epon sections (A–D). Specimen preparation: fixation-perfused specimens (A–L). Scale bars: A 5 25 mm; B–I 5 10 mm; J–L 5 50 mm.

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TABLE 4 Quantitative Analyses of Vessel Parameters in Specimens Perfused with FITC-Labeled Lycoperiscon Esculentum Lectin at 16 Months* Parameter per 340 high-power field Mean number of vessels Mean % area occupied by vessels Mean vessel diameter: direct measure of 5 representative vessels per field in mm Mean vessel diameter: image analysis of all vessels in mm Number of branches

Ad-VEGF-A164-treated striatum

Ad-lacZ-treated striatum

Normal striatum

p value†

22.3 (SE 0.4) 3.38 (SE 0.08) 5.42 (SE 0.05)

19.5 (SE 0.5) 1.91 (SE 0.08) 4.46 (0.05)

19.4 (SE 0.3) 2.15 (SE 0.05) 4.45 (SE 0.04)

,0.001 ,0.001 ,0.001

5.71 (SE 0.05)

4.56 (SE 0.06)

4.86 (SE 0.04)

,0.001

0.82 (SE 0.06)

0.62 (SE 0.07)

0.75 (SE 0.05)

0.103

* Data was obtained from 4 animals for each group. For each animal, 6 sections were analyzed with images in each taken at 340 magnification over 10 contiguous fields totaling 0.65 mm2 centered within the reactive site. † Statistical comparison is made between Ad-VEGF-A- and Ad-lacZ-treated striatum.

DISCUSSION The end result of Ad-VEGF-A164 angiogenesis in brain was the formation of a stable daughter neovasculature. Our studies demonstrate that daughter vessels evolved from a sequence of vascular changes in nascent vessels of the brain. Greatly enlarged mother vessels and complex glomeruloid bodies were identified as key intermediates leading to daughter vessel formation. Mother vessels likely characterize the enlarged vessels observed in previous studies that examined VEGF-A minipump and Ad-VEGF-A treatment at short time points (18–20). Our studies significantly extend the mechanisms, temporal profile, and morphological study of VEGF-A angiogenesis in brain (21). We found that mother vessels subsequently transformed into glomeruloid bodies through proliferation of endothelial cells and pericytes. In this process, small vessel lumens were created within glomeruloid bodies that thereafter matured into stable daughter vessels. While steps of mother vessel formation and glomeruloid body organization parallel and augment those previously observed in non-neural tissue, our current study exemplifies the tissue specific nature of the resultant angiogenic response (16, 35). Compared to skin wherein daughter vessels resolve over a period of weeks, our findings establish evidence of small daughter vessel perpetuity in the absence of ongoing exogenous VEGFA stimulation (35). Although this may appear to accomplish a long-sought goal of angiogenesis research, accompanying vascular hyperpermeability, cerebral edema, and J Neuropathol Exp Neurol, Vol 63, August, 2004

hemorrhage manifest the potential hazards associated with direct therapeutic application of VEGF-A for vessel augmentation in brain. Our data support a model in which daughter vessels evolved during glomeruloid body apoptosis and involution as a result of the balance between forces of vessel regression and stabilization. In terms of vessel regression, decreasing levels of VEGF-A mRNA correlated temporally with glomeruloid body involution. Strong expression of Ang2 mRNA was observed within glomeruloid bodies, consistent with the role of this cytokine in vessel destabilization and the remodeling phases of angiogenesis (44). More specifically, Ang2 has been shown to incite endothelial cell death and regression in low VEGF-A environments and may act cooperatively with declining VEGF-A levels to signal glomeruloid body involution (45). The small vessels within glomeruloid bodies survived. Pericytes have been shown to stabilize vessels, leading us to postulate that the admixture of pericytes and small vessel lumens within glomeruloid bodies facilitated the stabilization of evolving daughter vessels during glomeruloid body involution (46). We closely examined our studies for evidence of daughter vessel formation from other possible mechanisms. Daughter vessels did not arise as a result of sprouting angiogenesis, wherein vessels branch from a pre-existing vessel, as we observed an absence of abluminal endothelial cell migration towards the neural parenchyma. We also refuted the possibility that daughter vessels arise from unmasking of a pre-existing reserve capillary network. Controversial, the existence in brain of a nonperfused reserve network responsive to physiological stimuli has been debated (47–49). Our findings of daughter vessels amidst involuting glomeruloid bodies demonstrate that daughter vessels are a direct result of VEGFA-induced angiogenesis and argue that they do not result from reperfusion of a reserve vasculature. Several factors may have contributed to the long-term survival of daughter vessels in the absence of ongoing


7E–H). Some daughter vessels were situated close to one another and approximated the density of their parent glomeruloid bodies, while others masked their heritage and were distributed in a more dispersed pattern. Overall, vascular density at 16 months following injection of AdVEGF-A164, measured as a percentage of area within the angiogenic reaction site, showed a statistically significant .1.5-fold increase compared to Ad-lacZ-treated and normal striatum (Fig. 7M; Table 4).


become secondarily muscularized due to flow- and pressure-related change as an arterial angiogenic response induced by VEGF-A. Our identification of the vascular intermediates leading to daughter vessel formation models angiogenesis associated with a number of cerebral disease processes. Firstly, mother vessels together with bridged vascular structures and caverns resemble the thin-walled, endothelial lined cavities characteristic of cavernous malformations (64–67). Importantly, caverns appear unique to the cerebrum, as they are not a component of the VEGF-Ainduced angiogenic response in other tissues (16). Furthermore, multiple cavernous lesions have a predilection for the central nervous system (68). Together, these findings and data may suggest that the neural environment has a particular predisposition to foster cavernous vascular structures in response to angiogenic stimuli. Secondly, glomeruloid bodies represent the vascular signature of glioblastoma multiforme tumors (9, 10, 69). Our studies show that glomeruloid body formation was initiated in the walls of mother vessels. VEGF-A appears necessary for glomeruloid body formation, as strong VEGF-A mRNA signal localized to the epicenter of glomeruloid bodies. Furthermore, glomeruloid bodies are closely associated with glioblastoma multiforme tumors and yet rarely observed in other tumors. This may reflect the high levels of VEGF-A expressed by glioma tumors (70). Lastly, daughter vessels, the final result of VEGFA-induced cerebral angiogenesis, may model the small vessels formed during the angiogenic response to head injury and stroke (2, 4, 8). Taken together, our findings may provide a unifying mechanistic understanding of angiogenesis in cerebral disease. Mother vessels and related bridged and cavernous structures model vascular lesions such as cavernous malformations and appear to comprise an early and immature angiogenic response to VEGF-A. These lesions may be dependent upon ongoing VEGF-A expression for their survival. We hypothesize that high levels of VEGFA may be necessary to effect mother vessel to glomeruloid body transformation as characteristic of glioblastoma tumors, and that transient expression of VEGF-A may invoke but not sustain glomeruloid bodies. Subsequent involution of glomeruloid bodies to daughter vessels, as seen in our Ad-VEGF-A164 studies, may account for angiogenic vessels formed as a reactive response to injury. In conclusion, Ad-VEGF-A164 inoculation of adult murine striatum engendered a daughter neovasculature that persisted long-term in the absence of ongoing exogenous VEGF-A164 stimulation. Furthermore, our studies establish a mechanistic model for understanding angiogenesis associated with vascular malformations, glioblastoma multiforme tumors, and reactive angiogenesis to injury. J Neuropathol Exp Neurol, Vol 63, August, 2004


exogenous VEGF-A expression. First, we found that daughter vessels were richly invested by pericytes that have been shown to enable vessel stabilization in the absence of VEGF-A (46). Pericytes have been demonstrated to express Ang1, an important factor in vessel stabilization that may act to inhibit endothelial cell apoptosis via a PI3kinase/AKT-dependent pathway (50–53). We identified expression of Tie2 mRNA, but were unable to detect Ang1 mRNA, in evolving daughter vessels. Others have observed Tie2 mRNA and, more recently, Ang1 expression in the quiescent cerebral vasculature suggesting that together, they may play a role in vessel maintenance (54–56). Secondly, alterations in the composition of the extracellular matrix may also contribute to the long-term stability of daughter vessels. We found collagen, not a normal component of the neuropil, in the extracellular matrix surrounding daughter vessels. In experimental models of spinal cord injury, collagen scar formation is associated with upregulation of transforming growth factor b2 (TGF-b2) by neovascular endothelial cells (57). In vitro, TGF-b has been shown to stabilize endothelial and pericyte interactions (58). Lastly, constituents of the basement membrane may act to stabilize daughter vessels. Collagen IV and laminin have been shown to foster neovessel survival in an aortic ring model of angiogenesis implicating integrin receptor signaling and adhesion-dependent survival mechanisms (59, 60). Strong expression of collagen IV and laminin may thus further contribute to the long-term stabilization of daughter vessels. In normal adult murine brain, VEGF-A mRNA expression has been shown to be present in the choroid plexus, ependymal lining, as well as in occasional scattered cells in cortex (13, 41). Thus, we cannot exclude the further possibility that low levels of endogenous VEGF-A expression also contribute to the stabilization of daughter vessels. In addition to being stabilized, daughter vessels were perfused to 16 months as evidenced by staining of the endothelium following circulation of intravenous Lycoperiscon esculentum lectin. Furthermore, we observed enlarged vessels draining the angiogenic site and our studies identified thick, muscularized vessels with asmooth muscle actin immunoreactivity distributed about the periphery of the angiogenic reaction. Many of these vessels were remote to the area of VEGF-A expression. We interpret these vessels to represent normal cerebral vessels subjected to increased flow- or pressure-related vascular change induced by the daughter neovasculature (61–63). Indeed, we observed a local 1.5-fold increase in vessel density and expect that the draining vessels would have to accommodate the corresponding increase in blood flow. Because of their prominence, muscularized vessels are readily identified in long-term Ad-VEGF-A164treated specimens. However, it is important to distinguish the true neovasculature and not to interpret vessels that

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Together, we have identified a framework for future investigation of Ad-VEGF-A164-induced neovessels to enable therapeutic vessel modulation for a number of disease processes in the central nervous system. ACKNOWLEDGMENTS We thank Dr. James Kirby for his thoughtful critique and suggestions in the preparation of this manuscript.

REFERENCES



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