1 Regeneron Pharmaceuticals Incorporated, 777 Old Saw Mill River Road,
Tarrytown, NY 10591, USA
2 Cardiovascular Research Institute, UCSF Comprehensive Cancer Center, and
Department of Anatomy, University of California, San Francisco, CA 94143-0452,
USA
* Author for correspondence (e-mail: gavin.thurston{at}regeneron.com)
Accepted 3 May 2005
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SUMMARY |
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Key words: Angiogenesis, TIE2 receptor, Endothelial cells, Venules
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Introduction |
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Most studies of blood vessel development have focused on the sprouting
phase of angiogenesis. The best-characterized angiogenic agent, vascular
endothelial growth factor (VEGF), plays a key role in the formation of a
primitive vascular plexus by promoting endothelial cell proliferation,
sprouting and initial tube formation
(Nehls et al., 1994;
Wilting et al., 1993
). Genetic
deletion studies have confirmed that VEGF is required for these developmental
processes in vivo (Carmeliet et al.,
1996
; Ferrara et al.,
1996
). Furthermore, even when VEGF is administered to adult
animals, it retains its ability to induce sprouting and the formation of new
vessels in normal tissues (Pettersson et
al., 2000
; Springer et al.,
1998
; Springer et al.,
2000
). Reciprocally, inhibition of VEGF signaling potently
inhibits angiogenic sprouting in many situations of normal or pathological
angiogenesis, such as that associated with tumors
(Asano et al., 1995
;
Holash et al., 2002
;
Kim et al., 1993
;
Wood et al., 2000
).
In contrast to abundant data indicating that VEGF is a crucial mediator of
sprouting angiogenesis, much less is known about which factors may be involved
in subsequently regulating the diameter and remodeling the structure of the
primitive vessels, thereby allowing them to become specialized for their
position in the vascular network. On the arterial side of the circulation the
remodeling process is known as arterialization, and appears to involve the
interactions of flow, pressure, and agents such as platelet derived growth
factor B (PDGFB) that promote the interaction of endothelial tubes with smooth
muscle cells (Hellstrom et al.,
1999; Lindahl et al.,
1997
). An analogous process presumably occurs on the venous side,
although even less is known about this process.
Gene deletion studies have shown that vascular-specific growth factors or
receptors, such as angiopoietin 1 (Davis et
al., 1996; Dumont et al.,
1994
; Sato et al.,
1995
; Suri et al.,
1996
), angiopoietin 2 (Gale et
al., 2002
) and ephrin B2 (Shin
et al., 2001
; Wang et al.,
1998
), act later than VEGF during embryonic vascular remodeling
and maturation. Genetic deletion of these growth factors or their receptors
results in embryonic lethality and/or defects in vascular remodeling
subsequent to the key initial vasculogenic and angiogenic steps that are
dependent on VEGF. Although the precise roles of these other factors in
regulating the vasculature have yet to be clearly defined, gene deletion
studies suggest that angiopoietin 1 (ANG1; ANGPT1 Mouse Genome
Informatics/Human Gene Nomenclature Database) and its receptor TIE2 (TEK
Mouse Genome Informatics/Human Gene Nomenclature Database) are
involved in establishing a hierarchy of vessels, and are required for the
normal interactions between perivascular cells and endothelial cells
(Dumont et al., 1994
;
Sato et al., 1995
;
Suri et al., 1996
). While
potent inhibitors are not yet available for the ANG1/TIE2 system, treatment of
adult mice with soluble TIE2 receptor (a weak inhibitor) does not cause
obvious vascular changes in normal organs
(Lin et al., 1997
) (G.T.,
D.J.-G. and Q.W., unpublished).
Studies using overexpression systems have shed some light on the functions
of the ANG1/TIE2 signaling system in vivo, but have also raised key questions.
Constitutive transgenic overexpression of angiopoietin 1 in the skin of mice
(K14-ANG1 mice), starting in the early embryo, results in a dramatically
reddened appearance due to increased numbers of enlarged dermal microvessels
(Suri et al., 1998;
Thurston et al., 1999
). These
vessels are also resistant to leakage induced by VEGF or inflammatory agents
(Thurston et al., 1999
).
However, angiopoietin 1 treatment of adult mice does not change the morphology
of the skin vessels nor make the mice red, although it does make the dermal
blood vessels resistant to plasma leakage
(Thurston et al., 2000b
).
In contrast to the effects in adult mice, we now report that systemic angiopoietin 1 treatment of neonatal mice and rats results in conspicuously reddened pups containing enlarged blood vessels in the skin and in numerous other organs. The dramatic increases in vessel diameter are apparently caused by endothelial cell proliferation in the absence of vessel sprouting. Notably, the enlargement is largely restricted to the venous side of the microvasculature, and is not accompanied by changes in the number or pattern of vessels. By postnatal day (P) 30, vessels in most organs no longer enlarge in response to angiopoietin 1, indicating the passing of a critical period for vessel plasticity. VEGF-dependency of the vasculature in some organs of neonatal mice corresponds to a similar critical period of vessel plasticity. These findings show that angiopoietin 1 has a potentially unique role among the vascular growth factors, by acting to specifically increase blood vessel diameters without inducing sprouting, and also reveal a window of vascular plasticity in neonatal mice for multiple growth factors in multiple organs.
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Materials and methods |
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Angiopoietin 1 and VEGF-Trap treatments
For treatment of mice aged P7 to P49, pups from litters of C57BL/6 mice
(Taconic Laboratories, NY) were randomized and injected intraperitoneally with
ANG14FD (20, 50 or 200 µg) daily, or with PBS for controls. Rats
(Sprague-Dawley, Taconic) aged P7 were injected either daily or every other
day with ANG14FD [200 or 500 µg intraperitoneally (ip)]. Mice
treated with VEGF-Trap were given ip injections of 25 mg/kg every second day.
For experiments with adult mice, male pathogen-free FVB/N, C57BL/6 or CD-1
nude mice (Taconic Laboratories, NY, or Charles River, Hollister, CA) were
used at age 7-12 weeks. For adenovirus treatments, mice were anesthetized with
ketamine/xylazine, then injected into the jugular vein with 109
plaque forming units (pfu) of adenovirus encoding ANG1* diluted to 150 µl
in sterile saline (Thurston et al.,
2000b). Injection of Ad-ANG1* via the tail vein of mice had very
similar effects on blood vessel morphology to jugular vein injection. In some
mice, adenovirus was given locally, either via injections into the ear skin (2
x108 pfu in 5-8 µl) or intranasally (1
x109 pfu in 50 µl). Systemic VEGF was not used because of
toxicity (Thurston et al.,
2000b
).
ELISA for ANG14FD and ANG1* in serum
To confirm delivery of angiopoietin reagents and measure circulating
levels, blood (0.2 ml) was withdrawn from the right ventricle of anesthetized
mice and rats immediately prior to perfusion, centrifuged to obtain plasma,
and frozen until analysis. ANG1* and ANG14FD were measured by
ELISA, using recombinant TIE2 for capture, and an antibody against the N
terminus of ANG2 (which is present in ANG1*) or the human Fc domain of IgG (in
ANG14FD) to report.
Staining and measurement of neonatal vessels
Tissues from mouse and rat pups were harvested and immersion-fixed in
paraformaldehyde (1% in PBS, pH 7.4) for 1 hour to overnight. Tissues were
permeabilized with 0.3% Triton-X100, stained as wholemounts with hamster
anti-mouse PECAM antibody (Serotec, used at 1:500) or mouse RECA antibody
(Serotec), and, for mice, Cy3-labeled mouse anti- smooth muscle cell
actin antibody (Sigma, used at 1:500), followed by FITC-labeled goat
anti-hamster or goat anti-mouse antibodies (Jackson ImmunoResearch, West
Grove, PA; used at 1:500), and mounted in Vectashield. Staining blood vessels
with this method clearly labels the endothelial cells of the vessels, as well
as any endothelial sprouts that emanate from the vessels into the
interstitium. Confocal fluorescence images were collected using an inverted
confocal microscope (Leica) and a 40 x oil immersion lens. Measurements
of vessel diameter were performed on the tracheal vasculature
(McDonald, 1994
;
Thurston et al., 1998
).
Measurements were made on three tracheas per group on four representative
regions across the cartilaginous rings. Average vessel diameter was expressed
as mean±s.e.m.
BrdU labeling
A thymidine analog, 5-bromo-2'-deoxyuridine (BrdU; Sigma) was
injected intravenously (1 mg in 100 µl PBS) into mice. Three hours later,
mice were fixed by vascular perfusion of 1% paraformaldehyde in PBS. Tongues
were removed, washed in PBS, embedded in paraffin wax, cut into 8-µm-thick
sections and stained for BrdU (Staining Bulk Kit; Zymed Laboratories, San
Francisco, CA). Retinas were removed and stained whole, using Cy3-labeled
secondary antibodies and no counterstaining, or were counterstained with
FITC-labeled lectin (GSL I isolectin B4, Vector Laboratories). To
quantify the number of nuclei labeled by BrdU, low magnification images of
whole retinas were recorded digitally, and software was used to demarcate the
inner half of the retina. Labeled nuclei were counted in the inner half of the
retinas for three mice per group, and the average number of labeled nuclei was
expressed as mean±s.e.m.
Western blots for phosphotyrosine
One day after the final ip injection of ANG14FD or
ANG12FD, mice were sacrificed, and the trachea and lungs were
removed and rapidly frozen. Tissue was homogenized in buffer containing
protease and phosphatase inhibitors plus 0.1% NP40, and protein levels in each
homogenate were assessed using a micro-BCA assay (Pierce, Rockford, IL). TIE2
was immunoprecipitated overnight from 1 mg of lung lysate with 2 µg of the
anti-TIE2 antibody mab33 (K. Peters, Proctor and Gamble) and protein G beads
(Pharmacia), electrophoresed under reducing conditions, and transferred to
Immobilon-P membranes (PVDF; Owl Separation Systems, Portsmouth, NH).
Phosphotyrosine immunoreactivity was detected using an anti-phosphotyrosine
antibody (Upstate Biotechnology, Lake Placid, NY) and an HRP-conjugated goat
anti-mouse secondary antibody (Promega, Madison, WI), followed by
chemiluminescent detection (Amersham, Arlington Heights, IL). Total TIE2
protein was detected by stripping the blots and re-probing with the mab33
anti-TIE2 antibody.
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Results |
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Associated with the angiopoietin 1-induced reddening, we observed enlargement of the blood vessels of numerous organs, such as the trachea (Fig. 2A,B), tongue (Fig. 2C,D), diaphragm (Fig. 2E,F), retina (Fig. 2G,H), skin of the snout and mucosa of the bladder (data not shown). Of the tissues examined [ear skin, snout skin, tongue (mucosal, muscle), eye (cornea, retina), pancreas (islets, acinar), small intestine (mucosal, submucosal, muscular), bladder (mucosal), trachea (mucosal), diaphragm (central tendon, muscular), esophagus (muscular), kidney (glomerulus, medulla)], the vasculature of the brain and intestine (data not shown) was not notably enlarged. In the affected organs, the enlarged vessels were obvious in whole-mount views, as well as in thick and thin sections (Fig. 2, and results not shown). Strikingly, the vessel enlargement was largely restricted to the venous side of the circulation, including venular capillaries, postcapillary venules and collecting venules (arrows in Fig. 2), whereas arterioles (arrowheads in Fig. 2) were not enlarged by angiopoietin 1 treatment.
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Angiopoietin 1-induced vessel enlargement is associated with endothelial cell proliferation
To determine whether the vessel enlargement induced by angiopoietin 1 was a
result of vasodilation or remodeling (i.e. increased vessel size without more
endothelial cells or increased the number of endothelial cells), we determined
whether the vessels contained more endothelial cells and whether the
enlargement was accompanied by endothelial cell proliferation. The number of
endothelial cells was estimated in tracheal wholemounts stained for PECAM
immunoreactivity, which outlines the endothelial cell junctions (see Fig. S1
in the supplementary material). Normal postcapillary venules in the airways
were lined circumferentially by one or two endothelial cells. By comparison,
after angiopoietin 1 treatment, the enlarged vessels had three or more
endothelial cells lining their circumference (see Fig. S1 in the supplementary
material). In addition, BrdU-labeled endothelial cells were more abundant in
enlarged vessels of the retina (Fig.
4A,B) and tongue (see Fig. S1) in mice treated with angiopoietin 1
than in the corresponding vessels from control pups. The increased numbers of
BrdU-labeled endothelial cells were clearly preferentially distributed on the
venous side of the circulation (Fig.
4C,D, arrows). Because the retina was amenable to whole-mount BrdU
staining, we quantified BrdU labeling in retinas from mouse pups, and found
that angiopoietin 1 treatment resulted in 4-fold more BrdU-labeled endothelial
cells in the retina, when compared with controls
(Fig. 4E). Thus, vessel
enlargement induced by ANG1 results from increased numbers of endothelial
cells and endothelial cell proliferation, and is not due to vasodilation
alone.
|
The tracheal vessels in adult mice were also responsive to local delivery of angiopoietin 1. When delivered intranasally into adult mice, adenovirus encoding ANG1* resulted in infection of the airway epithelium and caused enlargement of the tracheal venules without sprouting. Intranasal delivery of control adenoviruses encoding green fluorescent protein did not cause changes in vessel morphology (Fig. 5I,J). By contrast, the same angiopoietin-encoding adenovirus injected into the ear skin did not induce enlargement of the skin microvessels (data not shown).
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Discussion |
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Despite previous reports that angiopoietin 1 might be able to induce vessel
sprouting and migration in vitro (Koblizek
et al., 1998), the data herein indicate that any such effects
would be minor when compared with the ability of angiopoietin 1 to regulate
vessel diameter. Thus, as previously suggested, VEGF and angiopoietin 1 are
complementary in their actions that is, while VEGF seems to promote
early phases of vasculogenesis and angiogenic sprouting, angiopoietin 1 seems
to be more relevant to the subsequent processes that regulate vessel size and
maturation. In addition, angiopoietin 1 is able to mediate its actions
independently of VEGF. Interestingly, although both factors have relatively
minor proliferative actions on cultured endothelial cells when compared with
other growth factors such as FGF, they both seem to be capable of promoting
endothelial proliferation in vivo, whereas the in vivo proliferative actions
of FGF remain to be validated.
|
The vessel enlargement induced by angiopoietin 1 appears to be rather
specific to the venous side of the microcirculation. Venules are specialized
functionally, morphologically and molecularly
(Thurston et al., 2000a).
Functionally, venules are most leaky to plasma proteins under baseline
conditions and are the site of inflammation-induced plasma leak
(Majno et al., 1969
). Venular
endothelial cells have a distinct molecular profile, including increased
expression of P-selectin, von Willebrand factor
(Thurston et al., 2000a
), and
receptors for inflammatory mediators
(Bowden et al., 1994
;
Heltianu et al., 1982
). In
addition, venules are the segment most likely to sprout during angiogenesis
(Folkman, 1982
;
Phillips et al., 1991
).
Previous studies have suggested that the venous side of the circulation may be
most responsive to, or dependent upon, angiopoietin 1 during development
(Loughna and Sato, 2001
;
Moyon et al., 2001
;
Thurston et al., 1999
). The
enlargement of the venules could be due to an abundance of TIE2 receptors on
the endothelial cells of venules, to increased accessibility to the abluminal
surface, to localized expression of angiopoietin 1, or to general plasticity
due to specialized pericytes and the basement membrane. Alternatively,
previous studies have noted that angiopoietin 2, which can act as an
antagonist of angiopoietin 1, is expressed in arterial smooth muscle cells,
with much weaker expression on the venous side
(Gale et al., 2002
;
Moyon et al., 2001
). Thus, the
arterial expression of angiopoietin 2 may act to inhibit angiopoietin 1 in
these vessels, and thus may explain why the venous side of the circulation is
more responsive to angiopoietin 1 stimulation.
Angiopoietin 1 appears to be able to regulate vessel size in most organs
only during a critical developmental window. This window coincides with a
period during which vessels in many organs are dependent on VEGF for survival.
Subsequent maturation of the vessels in many organs makes them less responsive
to vessel enlargement after angiopoietin 1 treatment and less dependent on
VEGF for survival. The evidence strongly suggests that these mature vessels do
not lose all responsiveness to angiopoietin 1, because angiopoietin 1
treatment of mature vessels results in a reduced plasma-leakage response
(Thurston et al., 2000b).
Thus, the developmental window appears to be a period when vessels maintain
plasticity to remodel morphologically in response to angiopoietin 1. By
comparison, even mature vessels appear to maintain plasticity to remodel in
response to VEGF, because robust angiogenesis occurs when exogenous VEGF is
applied to adult tissues that were not responsive to angiopoietin 1, for
example, the skin and the heart
(Pettersson et al., 2000
)
(data not shown).
So what reduces plasticity as vessels mature? Based on previous studies
(Benjamin et al., 1998;
Hirschi and D'Amore, 1997
), it
is possible that vessel maturation involves changes in the association of
endothelial cells with the surrounding perivascular support cells. Although
our preliminary data indicate that the neonatal vessels responsive to
angiopoietin 1 are already associated with perivascular cells, it is likely
that the interactions between endothelial cells and perivascular cells
continue to mature after initial investment. Indeed, even in adulthood, the
blood vessels of the airways continue to respond to angiopoietin 1 by
enlarging, although these vessels are covered by seemingly mature pericytes.
The response of the vasculature does not seem to depend on the route of
delivery, because the ear skin vessels are unresponsive to both local and
systemic delivery of ANG1, whereas the tracheal vessels are responsive to both
local and systemic delivery. Thus, the responsiveness of blood vessels to
angiopoietin 1 may be regulated by complex and poorly understood interactions
between endothelial and support cells, and/or basement membrane.
The ability to further characterize the maturity of blood vessels, the
nature of the interactions between endothelial cells and pericytes and
basement membrane, and the signals that underlie responsiveness to VEGF and
angiopoietin 1, seems likely to have important therapeutic implications. The
success of pro- and anti-angiogenic approaches in the clinic may well depend
on the ability to manipulate the state of vessel maturation, i.e. to revert
mature vessels to a more plastic state, or to induce plastic vessels to
mature. For example, in ischemic settings in which it is desirable to promote
vessel sprouting as well as increases in vessel size [such as of collaterals
feeding the ischemic tissue (Carmeliet,
2000)], one would want to induce a greater state of vessel
plasticity. Similarly, in tumors and other settings in which it may be
desirable to regress an existing vasculature, it may be useful to once again
induce vessel plasticity that would be associated with vascular instability
and increased responsiveness to VEGF blockade. Finding the key molecular and
cellular factors that regulate this plasticity switch may prove crucial to the
further development of pro- and anti-angiogenic therapies.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/14/3317/DC1
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