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Animal Models |
From the Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To elucidate the steps and mechanisms of pathological and physiological angiogenesis, we recently engineered adenoviral vectors to express angiogenic cytokines and have used these as vehicles for expressing these cytokines in mice and rats.1 Because of its prominent role in both angiogenesis and vasculogenesis, we chose vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) as the cytokine for initial study1,8,9 In the course of these studies, we noted that typical GB formed in intimate association with infected cells that expressed VPF/VEGF.1 Therefore, this system provided an excellent animal model for investigating the pathogenesis of GB formation. The present study was undertaken to elucidate the origins, composition, and fate of VPF/VEGF164-induced vascular GBs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four- to 6-week-old female athymic nude mice on two backgrounds were used for these studies with equivalent results: BALB/c ByJ hfh11nu (Jackson Laboratory, Bar Harbor, ME) and Nu/Nu (National Cancer Institute, Bethesda, MD).
Nonreplicating adenoviral vectors engineered to express murine VPF/VEGF164 (adeno-vpf/vegf164) or LacZ (adeno-lacZ) were prepared as previously described under the direction of Dr. Richard C. Mulligan in the Harvard Institute of Human Genetics.1 In brief, appropriate coding sequences were inserted into the pMDM transcriptional cassette consisting of a complete immediate early cytomegalovirus promoter and intron- and poly A-containing sequences derived from the human ß-globin gene.10,11 Vectors were purified using a double cesium chloride-banding procedure. Immediately before injection into mice, vectors were desalted using Quick Spin, High Capacity G-50 Sephadex columns (Boehringer Mannheim, Indianapolis, IN) and diluted in phosphate-buffered saline (PBS) and glycerol.
Vectors were injected intradermally into mouse ears with a 30-gauge needle. Virus (5 x 107 or 1 x 108 pfu) or vehicle alone (Hanks’ balanced salt solution) was administered in a volume of 10 µl. Animals were sacrificed by CO2 narcosis at different times (0, 4, 18, and 24 hours and 3, 7, 10, 14, 21, 28, 35, and 90 days). Ears were either embedded in OCT compound (Miles Diagnostics, Elkhart, IN) and frozen in liquid nitrogen for preparation of cryostat sections or were fixed in paraformaldehyde-glutaraldehyde and processed for 1-µm Epon sections12 or electron microscopy.13 All studies were performed under protocols approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee.
Antibodies and Other Reagents
Primary antibodies and their sources are listed in Table 1
.14-31
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All antibodies were diluted in solution 9 (PBS, pH 7.4, 0.1% bovine serum albumin, 150 mmol/L tranexamic acid, 20 µg/ml aprotinin (3 to 7 TIU/mg), 1.8 mmol/L ethylenediaminetetraacetic acid, and 2 mmol/L iodoacetic acid). Optimal antibody concentrations were determined by serial dilution.
Detection of Mouse Antigens with Mouse Monoclonal Antibodies
Six-µm serial cryostat sections were fixed in ice-cold acetone for 10 minutes and rinsed in PBS. IgG binding sites were blocked with normal rabbit serum diluted 1/5 in solution 9 further supplemented with 2% 3-omega fatty acid (Sigma) for 1 hour at room temperature. Complex formation between the primary and biotinylated secondary reagents was performed by appropriately diluting primary antibodies in solution 9 together with rabbit anti-mouse F(ab)2 IgG at a final concentration of 4 µg/ml for 1 hour at 37°C. Thereafter, to block residual binding sites for mouse IgG, mouse serum was added to a final concentration of 1/200, and incubated for 1 hour at 37°C. Sections were then incubated with the monoclonal antibody-F(ab)2-mouse serum complex for 1 hour at room temperature, rinsed, and incubated with 3% H2O2 in methanol for 5 minutes to deplete endogenous peroxidase. After rinsing, staining was performed with the avidin-biotin complex method, using the Vectastain ABC elite kit (Vector Laboratories) with diaminobenzidine as the peroxidase reporter. Sections were counterstained lightly with Mayer’s hematoxylin, dehydrated, and mounted in Permount.
Detection of Mouse Antigens with Rat or Rabbit Antibodies
Frozen sections were fixed, rinsed, and blocked as above. They were then incubated with primary antibody for 1 hour, rinsed in PBS, and depleted of endogenous peroxidase as above. After incubation with biotinylated rabbit anti-rat or goat anti-rabbit IgG, 7.5 µg/ml, for 30 minutes, staining was performed with the Vectastain ABC elite kit.
In Situ Hybridization
Tissues were fixed in RNase-free 4% paraformaldehyde in PBS, pH 7.4, for 4 hours at 4°C and were transferred to 30% sucrose in PBS overnight at 4°C before embedding in OCT compound. Cryostat sections were hybridized with antisense and sense (control), single-stranded, 35S-labeled RNA probes to murine VPF/VEGF, VEGFR-1, VEGFR-2, Ang-1, Ang-2, Tie-1, and Tie-2 as previously described.32,33
Double Staining and Confocal Imaging
One hundred-µm cryostat sections were fixed in 100% acetone at 4°C for 20 minutes and rehydrated in PBS containing 0.2% Tween 40. Sections were blocked with normal goat serum containing 2% 3-omega fatty acid for 1 hour at room temperature. Sections were then incubated for 2 hours with rabbit anti-mouse NG2, rinsed, and incubated with a second primary antibody, biotinylated rat anti-mouse CD31, for 2 hours. After rinsing, sections were incubated with avidin-coupled fluorescein isothiocyanate (FITC) for 2 hours. Sections were then washed 5x in distilled water and mounted with Vectashield (Vector Laboratories).
Perfusion Studies
To determine whether the GBs that had formed in mouse ears were perfused with blood, mice were injected intravenously with 4 mg of TRITC-dextran (MW, 70,000) and 4 mg of FITC-dextran (MW, 2,000,000) in 0.9% NaCl. Fifteen minutes later mice were sacrificed, ears were fixed in a 7:3 (vol:vol) mixture of absolute ethanol and 10% formalin for 4 hours at room temperature, and were then processed for paraffin embedding.12 Forty-micron optical sections were evaluated in a Bio-Rad MRC-1024 confocal microscope equipped with an argon/krypton laser. Sections were digitized, filtered with edge definition and median filters, and viewed as compiled images.
Alternatively, anesthetized mice were perfused through the left
ventricle with heparin-saline and then with 
20 ml of a 1:4 dilution
of Sumi black ink (Yasutomo and Co., South San Francisco, CA). Ears
were fixed in paraformaldehyde-glutaraldehyde, dehydrated in alcohols,
cleared in methyl salicylate, split, and mounted for microscopic
analysis in Permount.
Cell Proliferation
Mice were injected intravenously with 40 µCi of 3H-thymidine. Two hours later, ears were fixed and processed for 1-µm Epon sections and autoradiography.12
Statistical Analyses
Statistical analyses were performed with the Student’s t-test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adeno-vpf/vegf164 induced a characteristic
sequence of events in the ears of nude mice as previously
reported.1
Microvessels were rendered hyperpermeable to
plasma proteins with consequent extravasation of fibrinogen and
deposition of an extravascular fibrin-rich provisional matrix. In
addition, many microvessels enlarged greatly to become thin-walled,
pericyte-poor mother vessels (Figure 1, a–f
; Figure 2
, a and b; Figure 3
; Figure 4, b and d
;
and Figure 5, a and b
). Mother vessels were evident as
early as 18 hours and increased in size and number through 3 to 4
days, after which they evolved into vascular structures of several
different types, one of which was GB.
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, and Tables 1 and 2
). Primitive GBs arose in immediate proximity to
adeno-vpf/vegf164-infected cells that expressed
abundant VPF/VEGF mRNA (Figure 4; b, d, and e
). They were derived from
a rapidly dividing cell population such that on days 4 and 5,
respectively, 22 ± 14% and 18 ± 8% (mean ± SD)
labeled with 3H thymidine (Table 2)
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. They also stained more intensely for VEGFR-2 (Figure 1e)
, but less
intensely for VE-cadherin (not shown), than adjacent mother-vessel
endothelial cells that were not involved in GB formation. None stained
with pericyte (Figure 1f)
or macrophage (not shown) markers. Pericytes
were, however, identified immediately abluminal to developing GBs by
staining with antibodies to pericyte markers such as 
-smooth muscle
actin (-SMA) (Figure 1f)
. During the course of mother-vessel
formation, staining for basal lamina proteins was reduced focally and
in some cases circumferentially.1
However, by 3 to 4 days,
basal lamina staining had returned to normal and even higher than
normal levels (Figure 1g)
. Mature GBs (7 to 10 Days)
The cells comprising primitive GB continued to proliferate actively (eg, 14 ± 7% of cells labeled with 3H thymidine on day 8) and became organized into cell nests of increasing size. Whereas on day 4 after adeno-vpf/vegf164 injection the number of cells comprising GBs in random 1-µm sections was 21 ± 14, by day 8 that number had increased to 50 ± 23 (P = 0.0002).
As they increased in number, the cells comprising GBs projected into
the lumens of mother vessels and also extended outward into the
surrounding extravascular matrix (Figure 2, a to e)
. As a result, they
encroached on and compressed the mother vessels from which they had
arisen, eventually dividing their lumens into much smaller channels
that were only marginally perfused with blood (Figure 2, c and d
;
Figure 3, a–c
). By day 8 it was often difficult to tell that GBs had
arisen from mother vessels. However, in whole-mount preparations the
close relationship between mother vessels and GBs could still be
discerned (Figure 3, d–f)
. GBs were sometimes found to link adjacent
mother vessels (Figure 3f)
.
The great majority of cells comprising mature GBs continued to express
endothelial cell markers (Table 1)
; ie, they expressed VEGFR-1 and -2
mRNAs (Figure 5)
and CD-31 (Figure 6a)
and VEGFR-2 proteins (not shown), but not neuropilin (not shown).
However, cells having ultrastructural characteristics of pericytes
(Figures 7 and 8)
and staining for pericyte markers
(Figure 6c)
were now also present. By immunohistochemistry, these cells
were strongly positive for NG2, the murine analogue of the high
molecular weight melanoma-associated antigen, and the platelet-derived
growth factor (PDGF)-ß receptor. However, they expressed lower
concentrations of proteins associated with contractile functions
(-SMA (compare Figure 6, b and c
), calponin, desmin, and smooth
muscle myosin) than pericytes of surrounding, normal microvessels or of
microvessels of normal skin (not shown). By immunohistochemistry
pericytes appeared to be admixed randomly with endothelial cells.
However, by electron microscopy, it was clear that pericytes were
always situated peripherally to small cell clusters of endothelial
cells with small or no evident vascular lumens (Figures 7 and 8)
.
Occasional endothelial cell-pericyte contacts were identified by
electron microscopy (not shown). Monocytes and macrophages also began
to appear as determined by staining with antibodies to F4/80 (Figure 6d)
and HLA DR2 (not shown) and by electron microscopy (Figure 7
and
Figure 8
); macrophages tended to concentrate at the interface between
GBs and the surrounding connective tissue matrix (Figure 6d)
. Prominent
lipid-filled cells were often found adjacent to or centrally placed
within GBs (Figure 1b
; Figure 2, a, b, and e
; Figure 7
; and Figure 8
).
Occasional eosinophils and neutrophils were also present, but not
nerves or mast cells. As at earlier times, basal lamina proteins were
deposited between the cells comprising GBs in abnormal thickened
multilayers (Figure 6f
and Figure 8
, inset).
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By day 14 GBs began to devolve into a series of smaller
microvascular units, each comprised of centrally placed endothelial
cells surrounded by pericytes (Figure 9)
.
Endothelial cell-pericyte orientations now more clearly resembled those
of normal microvessels. Individual pericytes were at first commonly
positioned between adjacent clumps of endothelial cells, but,
throughout time, each endothelial cell cluster came to acquire its own
mantle of pericytes. Endothelial cells once again began to express VE
cadherin (not shown) and vascular lumens reappeared centrally in
endothelial cell clusters (Figure 2f
and Figure 9, c–e
). Concomitant
with these events, basal lamina proteins were deposited between
endothelial cells and pericytes in a distribution typical of normal
vessels (Figure 9, e and f)
. In this manner, individual,
normal-appearing microvessels were formed and began to spin off from
the residual GB mass (Figure 9, g–i)
. Macrophages decreased markedly
in number. GB devolution was accompanied by significant apoptosis,
evident on day 14 and maximal at day 21 (Figure 10)
. In parallel, numerous epidermal
cells underwent apoptosis and the epidermis, which had become
hyperplastic in the course of GB formation, gradually returned to
normal thickness.
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During this period, GBs disappeared altogether as distinct
entities and were replaced by multiple small daughter microvessels. As
newly formed microvessels formed and began to separate from GBs, a
subpopulation of pericytes appeared that, in addition to expressing the
PDGF-ß receptor (not shown) and NG2 (Figure 11b)
, also strongly expressed -SMA
(Figure 11a)
, 
-actin, desmin, smooth muscle myosin, and calponin
(not shown), a complement of proteins that comprise the cell’s
contractile machinery. Pericytes expressing this set of proteins were
observed at points of residual contact between adjacent developing
daughter vessels (Figure 11, a and b)
. Fibrillar collagen was also
deposited in these same locations (Figure 11c)
, progressively replacing
the fibrin provisional matrix and further separating individual
daughter microvessels from each other. Four to 5 weeks after injection
normal-appearing daughter capillaries and venules were evenly dispersed
(not shown), much as in normal ear skin.
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In situ hybridization was used to identify the cells
that were infected by adeno-vpf/vegf164 and to
investigate the kinetics of VPF/VEGF164
expression. From 18 hours to 4 days cells expressing abundant VPF/VEGF
mRNA were present in the dermis, especially in the deep dermis,
adjacent to the ear cartilage (Figure 4, a–e)
. Many of these heavily
labeled cells were in immediate proximity to developing or fully
developed mother vessels (Figure 4, b–e)
, ie, in a position normally
occupied by pericytes. However, the technologies we used did not allow
us to determine whether these
VPF/VEGF164-expressing cells were in fact
pericytes. At 3 days, strongly
VPF/VEGF164-expressing cells were found adjacent
to mother vessels in the immediate vicinity of developing GBs (Figure 4e)
, and at subsequent intervals strongly labeled cells were found in
the centers of mature GBs (Figure 4f)
. Curiously, prominent
lipid-filled cells were also commonly observed in the centers of mature
GBs (Figure 2, a, b, and e
, Figure 7
, and Figure 8
). Unfortunately, we
were unable to determine whether these lipid-filled cells were the
cells expressing large amounts of VPF/VEGF164
mRNA.
Because adenoviral vectors are not incorporated into chromosomal DNA,
the transgenes they introduce are only expressed for a limited period
of time. The numbers of VPF/VEGF mRNA-expressing cells and the
intensity of expression as judged by grains per cell remained constant
for 2 weeks, after which both the frequency and intensity of
labeling of individual cells declined gradually until by 21 days only
occasional, weakly positive cells remained (Figure 4, g–i)
.
VEGFR-1 and VEGFR-2 expression followed similar kinetics, becoming
detectable in mother vessel endothelial cells at 18 hours and more
strongly at 4 days (Figure 5)
. Thereafter, GBs continued positive for
the mRNAs of both receptors, declining after 2 weeks and falling to low
levels by 21 days.
Cells in GB expressed Ang-1 and Ang-2 mRNAs weakly from 10 to 21 days and expression declined thereafter. Tie-1 and Tie-2 mRNAs were weakly expressed in GBs as early as 3 days, strongly at 10 days, and weakly thereafter through 21 days.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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14 days, VPF/VEGF mRNA expression declined and,
concomitantly, GBs devolved by a process that involved both apoptosis
and reorganization of endothelial cells and pericytes into daughter
microvessels typical of those found in normal ear skin. The entire
sequence of events is diagrammed schematically in Figure 12
. Taken together, our experiments
provide the first animal model for generating vascular GBs, provide
important insights into the mechanisms responsible for GB formation and
devolution, and demonstrate that VPF/VEGF164 is
both sufficient for their generation and necessary for their
maintenance.
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-SMA.34,35
Thus, both pericytes and endothelium contribute importantly to the GBs
of high-grade human gliomas and to those induced here in normal mouse
tissues with an adenoviral vector. Whether the GBs that develop in
human glioblastomas also arise from mother vessels remains to be
determined. High concentrations of VPF/VEGF164 were apparently necessary to induce and sustain GBs. In our mouse model, GBs formed in immediate anatomical proximity to adeno-vpf/vegf164-infected cells that strongly expressed VPF/VEGF mRNA. Glioblastomas express larger amounts of VPF/VEGF than most other human tumors3,36,37 ; however, glioblastomas also express many other cytokines and growth factors that might reasonably be implicated in GB formation. Therefore, the finding that overexpression of VPF/VEGF164 alone leads to GB formation is significant and allows us to postulate that the GBs found in glioblastomas, in other human tumors, and in vascular malformations also result from local overexpression of VPF/VEGF. Further correlative evidence in support of VPF/VEGF’s role in the etiology of GB formation came from later events in our mouse adenoviral vector model. As VPF/VEGF164 expression declined, GBs began to devolve. Of course no such decline in VPF/VEGF expression would be expected in glioblastomas as these tumors continue to express VPF/VEGF and generate GBs throughout their course.
The cells that were first recognized as primitive GBs were located amid other endothelial cells lining mother vessels and expressed endothelial markers (CD31, VEGFR-1, VEGFR 2). Together these findings are consistent with an origin from pre-existing mother-vessel endothelium. However, the markers that these cells expressed are not entirely specific for endothelium and are also represented on hematopoietic cell precursors.38-40 Therefore, a blood or bone marrow source for some or all of these precursor cells cannot be excluded. Subsequently, pericytes also contributed to GBs and their origins were also not established with certainty. The likeliest possibility, however, is that they developed from mother-vessel pericytes because they first appeared admixed among endothelial cells; there was no evidence to suggest that they differentiated from primitive mesenchymal or other dermal cells.
The stimuli responsible for pericyte participation in GBs are uncertain. One possibility is that VPF/VEGF induced the proliferation of local pericytes by stimulating endothelial cells to express PDGF-B chain,41 a potent pericyte mitogen.42,43 PDGF BB is also chemotactic for pericytes44 and is thought to be important for pericyte differentiation and their coating of newly formed vessels during embryogenesis.44-47 However, recent findings raise the alternative possibility that VPF/VEGF acted directly on pericytes. In addition to its selective action on endothelial cells, VPF/VEGF interacts with macrophages and certain populations of smooth muscle cells that express biologically active VPF/VEGF receptors.8,9,48 VEGFR-1 has also been found to be expressed on cultured pericytes, and under certain conditions (hypoxia), VPF/VEGF is chemotactic for pericytes.49-51 These findings raise the possibility that VPF/VEGF stimulated pericyte migration and proliferation directly.
Beginning around day 14, GBs began a process of devolution that was characterized by significant apoptosis. Endothelial cells, pericytes, and macrophages all declined in number, paralleling a decline in local VPF/VEGF164 expression. VPF/VEGF is an important survival factor for vascular endothelium,47,52 particularly for immature vessels that have not acquired a pericyte coat. The endothelial cell apoptosis observed may therefore have resulted from a combination of decreased local VPF/VEGF and insufficient pericyte coating. Keratinocytes express VPF/VEGF,53 but have not been reported to express VPF/VEGF receptors. Most likely the apoptotic events observed reflect a series of complex paracrine relationships among the various cell types that were upset by decreased VPF/VEGF. Similar paracrine stimulatory loops are thought to maintain GBs in glioblastoma multiforme34,36,37,54,55 . These findings may have interesting implications for anti-angiogenesis therapies designed to suppress VPF/VEGF expression in glioblastomas. Our studies would suggest that decreased local VPF/VEGF expression would initiate GB devolution, and it is uncertain whether such devolution with replacement by normal microvessels would be of advantage to the tumor or to the host.
As GBs devolved, they were gradually replaced by normal-appearing
microvessels that at first were closely joined together and only later
separated from one another. A subpopulation of pericytes that expressed
proteins involved in contractile events may have had a role in
positioning and separating these newly formed daughter vessels from
each other. Pericytes that expressed -SMA, -actin, and so forth,
were strategically positioned at points of interconnection between
developing daughter vessels, suggesting a possible role in the final
separation of daughter vessels from each other. This would be
consistent with other examples in which pericytes expressing a similar
profile of contractile machinery have important roles in tissue
remodeling.56-58
In conclusion, injection of an adenoviral vector engineered to express
VPF/VEGF164 induced the formation of typical
vascular GBs in normal ear skin. These GBs developed in mother vessels
from recruitment and proliferation of endothelial cells and pericytes
in intimate contact with VPF/VEGF-expressing cells. As local VPF/VEGF
expression began to decline at 2 weeks, GBs gradually devolved, a
process characterized by apoptosis and reorganization of remaining
endothelial cells and pericytes into normal-appearing microvessels.
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Acknowledgements |
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Footnotes |
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Supported in part by U. S. Public Health Service grants CA-50453 and HL-59316 (to H. F. D.), AI-33372 and AI-44066 (to A. M. D.), by a contract from the National Foundation for Cancer Research (to H. F. D.), and by a grant from the Swedish Cancer Foundation, Konung Gustaf V:s 80 årsfond (to C. S.).
Present address of C. S.: Dept. of Medical Biochemistry and Microbiology, Uppsala University, BMC, Box 575, SE-751 23, Uppsala, Sweden.
Accepted for publication November 17, 2000.
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T. T. Rissanen, J. E. Markkanen, M. Gruchala, T. Heikura, A. Puranen, M. I. Kettunen, I. Kholova, R. A. Kauppinen, M. G. Achen, S. A. Stacker, et al. VEGF-D Is the Strongest Angiogenic and Lymphangiogenic Effector Among VEGFs Delivered Into Skeletal Muscle via Adenoviruses Circ. Res., May 30, 2003; 92(10): 1098 - 1106. [Abstract] [Full Text] [PDF]
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 J. Zhu, K. Motejlek, D. Wang, K. Zang, A. Schmidt, and L. F. Reichardt {beta}8 integrins are required for vascular morphogenesis in mouse embryos Development, March 8, 2003; 129(12): 2891 - 2903. [Abstract] [Full Text] [PDF]
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 J. A. Nagy, E. Vasile, D. Feng, C. Sundberg, L. F. Brown, M. J. Detmar, J. A. Lawitts, L. Benjamin, X. Tan, E. J. Manseau, et al. Vascular Permeability Factor/Vascular Endothelial Growth Factor Induces Lymphangiogenesis as well as Angiogenesis J. Exp. Med., December 2, 2002; 196(11): 1497 - 1506. [Abstract] [Full Text] [PDF]
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O. Straume, P. O. Chappuis, H. B. Salvesen, O. J. Halvorsen, S. A. Haukaas, J. R. Goffin, L. R. Begin, W. D. Foulkes, and L. A. Akslen Prognostic Importance of Glomeruloid Microvascular Proliferation Indicates an Aggressive Angiogenic Phenotype in Human Cancers Cancer Res., December 1, 2002; 62(23): 6808 - 6811. [Abstract] [Full Text] [PDF]
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 H. F. Dvorak Vascular Permeability Factor/Vascular Endothelial Growth Factor: A Critical Cytokine in Tumor Angiogenesis and a Potential Target for Diagnosis and Therapy J. Clin. Oncol., November 1, 2002; 20(21): 4368 - 4380. [Abstract] [Full Text] [PDF]
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 C. Ruhrberg, H. Gerhardt, M. Golding, R. Watson, S. Ioannidou, H. Fujisawa, C. Betsholtz, and D. T. Shima Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis Genes & Dev., October 15, 2002; 16(20): 2684 - 2698. [Abstract] [Full Text] [PDF]
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A. Saaristo, T. Veikkola, T. Tammela, B. Enholm, M. J. Karkkainen, K. Pajusola, H. Bueler, S. Yla-Herttuala, and K. Alitalo Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects J. Exp. Med., September 16, 2002; 196(6): 719 - 730. [Abstract] [Full Text] [PDF]
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D. M. McDonald and P. Baluk Significance of Blood Vessel Leakiness in Cancer Cancer Res., September 15, 2002; 62(18): 5381 - 5385. [Abstract] [Full Text] [PDF]
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 T. Morimoto, M. Aoyagi, M. Tamaki, Y. Yoshino, H. Hori, L. Duan, T. Yano, M. Shibata, K. Ohno, K. Hirakawa, et al. Increased Levels of Tissue Endostatin in Human Malignant Gliomas Clin. Cancer Res., September 1, 2002; 8(9): 2933 - 2938. [Abstract] [Full Text] [PDF]
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A. SAARISTO, T. VEIKKOLA, B. ENHOLM, M. HYTONEN, J. AROLA, K. PAJUSOLA, P. TURUNEN, M. JELTSCH, M. J. KARKKAINEN, D. KERJASCHKI, et al. Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes FASEB J, July 1, 2002; 16(9): 1041 - 1049. [Abstract] [Full Text] [PDF]
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 T. Odorisio, C. Schietroma, M. L. Zaccaria, F. Cianfarani, C. Tiveron, L. Tatangelo, C. M. Failla, and G. Zambruno Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability J. Cell Sci., June 15, 2002; 115(12): 2559 - 2567. [Abstract] [Full Text] [PDF]
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J. Sevigny, C. Sundberg, N. Braun, O. Guckelberger, E. Csizmadia, I. Qawi, M. Imai, H. Zimmermann, and S. C. Robson Differential catalytic properties and vascular topography of murine nucleoside triphosphate diphosphohydrolase 1 (NTPDase1) and NTPDase2 have implications for thromboregulation Blood, April 15, 2002; 99(8): 2801 - 2809. [Abstract] [Full Text] [PDF]
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 J.M. ARBEIT Quiescent Hypervascularity Mediated by Gain of HIF-1{alpha} Function Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 133 - 142. [Abstract] [PDF]
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J.A. NAGY, E. VASILE, D. FENG, C. SUNDBERG, L.F. BROWN, E.J. MANSEAU, A.M. DVORAK, and H.F. DVORAK VEGF-A Induces Angiogenesis, Arteriogenesis, Lymphangiogenesis, and Vascular Malformations Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 227 - 238. [Abstract] [PDF]
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C. Goepfert, C. Sundberg, J. Sevigny, K. Enjyoji, T. Hoshi, E. Csizmadia, and S. Robson Disordered Cellular Migration and Angiogenesis in cd39-Null Mice Circulation, December 18, 2001; 104(25): 3109 - 3115. [Abstract] [Full Text] [PDF]
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R. M. Tuder and N. F. Voelkel Plexiform Lesion in Severe Pulmonary Hypertension: Association with Glomeruloid Lesion Am. J. Pathol., July 1, 2001; 159(1): 382 - 383. [Full Text] [PDF]
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D. J. Brat and E. G. Van Meir Glomeruloid Microvascular Proliferation Orchestrated by VPF/VEGF : A New World of Angiogenesis Research Am. J. Pathol., March 1, 2001; 158(3): 789 - 796. [Full Text] [PDF]
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