Developmental Cell, Vol. 3, 411–423, September, 2002, Copyright 2002 by Cell Press
Angiopoietin-2 Is Required for Postnatal
Angiogenesis and Lymphatic Patterning, and Only
the Latter Role Is Rescued by Angiopoietin-1
lial growth factor (VEGF) family and Angiopoietin-1 (Car-
meliet et al., 2001; Eriksson and Alitalo, 1999; Ferrara,
tin-1 appear to work in complementary fashion during
early vascular development, with VEGF-A initiating vas-
cular formation (Carmeliet et al., 1996; Ferrara et al.,
1996) and Angiopoietin-1 promoting subsequent vascu-
lar remodeling, maturation, and stabilization, perhaps,
in part, by supporting interactions between endothelial
cells and surrounding support cells and matrix (Dumont
et al., 1994; Sato et al., 1995; Suri et al., 1996). Other
members of the VEGF family (i.e., VEGF-C and VEGF-D)
of the lymphatic vasculature, acting via VEGF recep-
tor-3 (VEGFR-3) (Jeltsch et al., 1997; Karkkainen et al.,
2000; Kukk et al., 1996; Makinen et al., 2001; Veikkola
have not been implicated in lymphatic development.
Previous descriptive studies suggest that the second
member of the Angiopoietin family, Angiopoietin-2, might
be a complex regulator of vascular remodeling that plays
a role in both vessel sprouting and vessel regression.
Supporting such roles for Angiopoietin-2, expression
analyses reveal that Angiopoietin-2 is rapidly induced,
together with VEGF, in adult settings of angiogenic
sprouting, whereas Angiopoietin-2 is induced in the ab-
sence of VEGF in settingsof vascular regression (Goede
Stratmann et al., 1998; Wiegand et al., 2000; Zagzag et
al., 1999). Consistent with a context-dependent role,
Angiopoietin-2 binds to the same endothelial-specific
receptor, Tie2, that is activated by Angiopoietin-1 but
has context-dependent effects on its activation. That is,
Angiopoietin-2 seems to activate Tie2 on some cells,
while it blocks Tie2 activation on other cells or under
different culture conditions (Davis et al., 1996; Maison-
pierre et al., 1997; Mochizuki et al., 2002; Teichert-Kulis-
zewska et al., 2001). It is not known whether Angiopoie-
tin-2 is acting as a Tie2 activator or blocker in any
specific in vivo setting.
In this study, we used a gene-targeting approach in
mice to examine the role of Angiopoietin-2 in vascular
development and function. We report that Angiopoie-
tin-2, unlike VEGF and Angiopoietin-1, is not requisite
during embryonic vascular development but, instead, is
necessary during subsequent postnatal vascular re-
modeling. Specifically, Angiopoietin-2 is absolutely re-
sion, which normally occur in coupled fashion during
thoroughly studied sites of postnatal vascular remodel-
ing (Alon et al., 1995; Benjamin et al., 1998; Hackett et
al., 2000; Ito and Yoshioka, 1999; Stone et al., 1995,
1996). Unexpectedly, we also found that deletion of An-
about the mechanism of action of Angiopoietin-2, we
generated mice in which the Angiopoietin-2 gene was
Nicholas W. Gale,1Gavin Thurston,1
Sean F. Hackett,2Roumiana Renard,1
Quan Wang,1Joyce McClain,1Cliff Martin,3
Charles Witte,3Marlys H. Witte,3David Jackson,4
Chitra Suri,1Peter A. Campochiaro,2
Stanley J. Wiegand,1and George D. Yancopoulos1,5
1Regeneron Pharmaceuticals, Inc.
777 Old Saw Mill River Road
Tarrytown, New York 10591
2Departments of Ophthalmology and Neuroscience
The Johns Hopkins University School of Medicine
600 North Wolfe Street
Baltimore, Maryland 21287
3University of Arizona College of Medicine
Department of Surgery
1501 Campbell Avenue
P.O. Box 245063
Tucson, Arizona 85724
4MRC Human Immunology Unit
Institute of Molecular Medicine
John Radcliffe Hospital
OX3 9DS Oxford
VEGF and Angiopoietin-1 requisitely collaborate dur-
ing blood vessel development. While Angiopoietin-1
obligately activates its Tie2 receptor, Angiopoietin-2
can activate Tie2 on some cells, while it blocks Tie2
activation on others. Our analysis of mice lacking An-
for embryonic vascular development but is requisite
mice lacking Angiopoietin-2 also exhibit major lym-
phatic vessel defects. Genetic rescue with Angiopoie-
tin-1 corrects the lymphatic, but not the angiogenesis,
defects, suggesting that Angiopoietin-2 acts as a Tie2
agonist in the former setting, but as an antagonist in
the latter setting. Our studies define a vascular growth
factor whose primary role is in postnatal angiogenic
remodeling and also demonstrate that members of
the VEGF and Angiopoietin families collaborate during
development of the lymphatic vasculature.
Only a small number of endothelial cell growth factors
have been confirmed as requisite for vascular develop-
ment, based on gene-targeting approaches. The list of
Figure 1. The Targeting Vector Designed to Substitute the LacZ Gene for Endogenous Angiopoietin-2 Gene and the Use of this Vector to
Generate Mice Carrying this Genetic Substitution
(A) The endogenous genetic locus containing the first coding exon (green cylinder) of Angiopoietin-2 (Ang2) is depicted, as is the targeting
vector designed to disrupt the first Angiopoietin-2 exon while inserting a promoterless LacZ gene under the control of the endogenous
Angiopoietin-2 promoter. The appropriately targeted Angiopoietin-2 locus is also shown, as are the predicted restriction fragment differences,
which should distinguish the targeted allele from the parental allele; correct targeting was determined using the indicated probes to detect
restriction fragment differences in BamH1-restricted DNA from ES cell clones (B, BamH1; S, Sac1; X, Xba1).
(B) Southern Blots reveal that correct gene targeting was achieved in ES clones 36 and 45, as evidenced by the presence of restriction
fragments diagnostic of the correctly targeted mutant allele (using the 3? probe in this example; see [A]) ([B], upper panel). Chimeric mice
generated from ES clones 36 and 45 were used to generate F1 progeny containing the mutant allele, which, in turn, were mated to yield wild-
type (?/?), heterozygous (?/LZ), and homozygous knockout mice (LZ/LZ), as confirmed by Southern blotting (using the 5? probe in this
example; see [A]) ([B], lower panel).
(C) Northern blots from a variety of tissues from newborn mice (hrt, heart; kid, kidney; lu, lung) were probed with a full-length Angiopoietin-2
cDNA to confirm that the amount of Angiopoietin-2 mRNA is reduced in Ang2?/LZheterozygotes and entirely missing in Ang2LZ/LZmice.
(D) Whole-mount views of the heart and major blood vessels in the thorax of E14.5 embryos heterozygous for the Angiopoietin-2 LacZ
substitution reveal ?-galactosidase (?-gal) activity in the aorta (A) and major vessels, but not in the heart proper (H).
(E) In contrast to Angiopoietin-2 expression, the heart, but not the blood vessels, exhibits ?-gal activity in mice in which the Angiopoietin-1
gene has been substituted with LacZ.
(F) Northern blots from total RNA prepared from P1 and 10-week-old wild-type mice confirm the fidelity of expression of the LacZ reporter
genes in the engineered strains and the complementarity of expression of Angiopoietin-1 and -2 in the heart and major arteries, both
embryonically and at early and late postnatal ages. Lungs have been removed in (D) and (E) to allow visualization of the heart and vessels.
replaced with cDNA encoding Angiopoietin-1. Surpris-
ingly, Angiopoietin-1 completely rescued the lymphatic
defects in mice lacking Angiopoietin-2, but not the de-
tin-2 acts as a Tie2 agonist in the former setting, but as
an antagonist in the latter setting. Thus, our studies
define a vascular growth factor that is dispensable for
embryonic angiogenesis but that is specifically required
for normal postnatal vascular remodeling. Furthermore,
our studies demonstrate that members of the VEGF and
Angiopoietin families work together not only during de-
velopment of the blood vasculature, but also during de-
velopment of the lymphatic vasculature.
Engineering of Mice Lacking Angiopoietin-2
To generate mice lacking Angiopoietin-2, we first con-
structed a targeting vector that replaced part of the
coding region of Angiopoietin-2 with the LacZ gene en-
coding ?-galactosidase (?-gal) (Figure 1A), with the in-
tention of creating a null allele that substituted ?-gal as
(referred to as an “Ang2-LacZ knockout allele,” Ang2LZ).
This targeting vector was used to alter the endogenous
Angiopoietin-2 allele in embryonic stem cells (Figure
1B, top panel), which were then used to generate mice
with reverse primers from either Angiopoietin-2 or Angiopoietin-1 to
detect either endogenous mRNA or inserted Angiopoietin-1 cDNA.
Adamis, A.P., Miller, J.W., Bernal, M.T., D’Amico, D.J., Folkman, J.,
Yeo, T.K., and Yeo, K.T. (1994). Increased vascular endothelial
growth factor levels in the vitreous of eyes with proliferative diabetic
retinopathy. Am. J. Ophthalmol. 118, 445–450.
Aiello, L.P., Avery, R.L., Arrigg, P.G., Keyt, B.A., Jampel, H.D., Shah,
S.T., Pasquale, L.R., Thieme, H., Iwamoto, M.A., Park, J.E., et al.
(1994). Vascular endothelial growth factor in ocular fluid of patients
with diabetic retinopathy and other retinal disorders. N. Engl. J.
Med. 331, 1480–1487.
Alon, T., Hemo, I., Itin, A., Pe’er, J., Stone, J., and Keshet, E. (1995).
Vascular endothelial growth factor acts as a survival factor for newly
turity. Nat. Med. 1, 1024–1028.
Banerji, S., Ni, J., Wang, S.X., Clasper, S., Su, J., Tammi, R., Jones,
glycoprotein, is a lymph-specific receptor for hyaluronan. J. Cell
Biol. 144, 789–801.
Benjamin, L.E., Hemo, I., and Keshet, E. (1998). A plasticity window
for blood vessel remodelling is defined by pericyte coverage of the
preformed endothelial network and is regulated by PDGF-B and
VEGF. Development 125, 1591–1598.
Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L.,
C., et al. (1996). Abnormal blood vessel development and lethality
in embryos lacking a single VEGF allele. Nature 380, 435–439.
Carmeliet, P., Moons, L., Luttun,A., Vincenti, V., Compernolle, V., De
Mol, M., Wu, Y., Bono, F., Devy, L., Beck, H., et al. (2001). Synergism
between vascular endothelial growth factor and placental growth
factor contributes to angiogenesis and plasma extravasation in
pathological conditions. Nat. Med. 7, 575–583.
J., Ryan, T., Bruno, J., Radjiewski, C., Maisonpierre, P.C., and Yan-
copoulos, G.D. (1996). Isolation of angiopoietin-1, a ligand for the
TIE2 receptor, by secretion-trap expression cloning. Cell 87, 1161–
DeChiara, T.M., Bowen, D.C., Valenzuela, D.M., Simmons, M.V.,
Poueymirou, W.T., Thomas, S., Kinetz, E., Compton, D.L., Rojas, E.,
Park, J.S., et al. (1996). The receptor tyrosine kinase MuSK is re-
quired for neuromuscular junction formation in vivo. Cell 85,
Dumont, D.J., Gradwohl, G., Fong, G.-H., Puri, M.C., Gerstenstein,
M., Auerbach,A., andBreitman, M.L.(1994). Dominant-negativeand
targeted null mutations in the endothelial receptor tyrosine kinase,
tek, reveal a critical role in vasculogenesis of the embryo. Genes
Dev. 8, 1897–1909.
Eriksson, U., and Alitalo, K. (1999). Structure, expression and recep-
tor-binding properties of novel vascular endothelial growth factors.
Curr. Top. Microbiol. Immunol. 237, 41–57.
Ferrara, N.(1999). Vascularendothelial growth factor:molecular and
biological aspects. Curr. Top. Microbiol. Immunol. 237, 1–30.
Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., O’Shea,
K.S., Powell-Braxton, L., Hillan, K.J., and Moore, M.W. (1996). Het-
erozygous embryonic lethality induced by targeted inactivation of
the VEGF gene. Nature 380, 439–442.
Gale, N.W., Baluk, P., Pan, L., Kwan, M., Holash, J., DeChiara, T.M.,
McDonald, D.M., and Yancopoulos, G.D. (2000). EphrinB2 selec-
with expression in both endothelial and smooth muscle cells. Dev.
Biol. 230, 151–160.
Goede, V., Schmidt, T., Kimmina, S., Kozian, D., and Augustin, H.G.
(1998). Analysis of blood vessel maturation processes during cyclic
ovarian angiogenesis. Lab. Invest. 78, 1385–1394.
Hackett, S.F., Ozaki, H., Strauss, R.W., Wahlin, K., Suri, C., Maison-
pierre, P., Yancopoulos, G., and Campochiaro, P.A. (2000). Angio-
poietin 2 expression in the retina: upregulation during physiologic
and pathologic neovascularization. J. Cell. Physiol. 184, 275–284.
Holash, J., Maisonpierre, P.C., Compton, D., Boland, P., Alexander,
Processing of Eye Tissue
Eyes werecollected fromgenetically modifiedand controllittermate
mice at ages P1–P10. One eye from each animal was fixed in 4%
paraformaldehyde, and the eye cups were processed for visualiza-
tion of ?-galactosidase activity (?-gal) as described previously (Gale
et al., 2000). The tissue was postfixed in 4% paraformaldehyde
overnight at 4?C, cryoprotected, and embedded in O.C.T. media
(Sakura FineTek, Torrance, CA). Series of 10 ?m were then cut, and
alternate series were immunostained for PECAM (platelet endothe-
lial-cell adhesion molecule) to visualize Angiopoietin-2-driven ?-gal
expression and vascular endothelial cellsin the same sections. Sec-
tions were pretreated for 20 min in 100 mM NaIO4to inhibit endoge-
nous peroxidase, rinsed with TBS, and permeabilized for 1 hr in 1%
BSA, 2% Rabbit serum, and 0.4% Triton-X. Sections were then
incubated overnight at 4?C in anti-PECAM (PharMingen; 1:150) fol-
lowed by applications of a biotinylated secondary antibody (rabbit
Peroxidase complex (Vectastain ABC kit; Vector Laboratories).
ine-peroxide solution containing nickel ammonium sulfate to yield
a black reaction product. The remaining eye from each animal was
embedded in paraffin, and sections were stained with hematoxylin
and eosin. In situ hybridization for localization of Angiopoietin-2
was performed on 10 ?m frozen sections of retina as described
(Maisonpierre et al., 1997). The pattern of expression of Angiopoie-
tin-2 mRNA in the retinas of mice heterozygous of Angiopoietin-2
deletion/LacZ substitution was identical to that seen in the eyes of
wild-type mice and also closely matched the pattern of ?-gal stain-
ing, thereby confirming the fidelity of the LacZ expression system.
Staining Whole-Mount and Thick Tissues with LYVE-1,
PECAM, and SMA Antibodies
Intestine, ear skin, and other tissues were collected and put into fix
(1% paraformaldehyde in PBS [pH 7.4]) for ?1 hr and then washed
with PBS. Some tissues were processed as whole mounts (e.g., ear
skin and intestine), whereas other tissues and parts of the intestine
were embedded in warmed (40?C) low-melting point agarose (FMC)
inPBS. Transversesectionsof intestinewerecut(100 ?mthickness)
with a Vibratome. Tissue was blocked (3% goat serum in PBS plus
0.3% Triton X-100, 2 hr) and then stained with rabbit anti-LYVE-1
(1:2000) and hamster anti-PECAM (1:500; Serotec) in PBS plus 0.3%
Triton X-100, followed by FITC goat anti-hamster (1:500; Jackson
Immunoresearch) plus Cy3 goat anti-rabbit (1:500; Jackson Immu-
noresearch) in PBS plus 0.3% Triton X-100. Alternatively, some tis-
sues were stained with Cy3-labeled anti-? smooth muscle actin
antibodies (1:500; Sigma). Tissues were mounted in Vectashield
(Vector Laboratories) and viewed with a confocal microscope
(Leica). Alternatively, to visualize anti-LYVE-1 staining with light mi-
croscopy, tissues were fixed and dehydrated through 25%, 50%,
75%, and 100% MeOH. Tissues were then bleached in 5% H2O2/
MeOH (5 hr), rinsed twice with 100% MeOH, rehydrated through
75%, 50%, and 25% MeOH/PBS, and blocked in blocking solution
(0.5% BSA and 0.1% TX-100 in PBS for 1 hr). Primary antibody was
incubated as above in blocking solution overnight at 4?C, washed
extensively with PBT (0.2% BSA and 0.1% TX-100 in PBS), incu-
bated in secondary antibody (goat anti-rabbit-biotin, Vector BA-
1000, 1:1500 dilution) in blocking solution overnight at 4?C. Tissues
were then washed extensively with PBT, incubated in avidin:bio-
tin:peroxidase complex (Vector Elite PK-6100, 1:2000 dilution) in
blocking solution overnight at 4?C, washed extensively, and equili-
brated in DAB developing buffer.
We thank Virginia Hughes and Mary Simmons for animal husbandry,
Li Pan and Danielle Jean-Guillaume for technical assistance, and
Scott Staton for imaging and graphics.
Received: October 15, 2001
Revised: June 14, 2002
Ang2 in Angiogenesis and Lymphatic Patterning
C.R., Zagzag, D., Yancopoulos, G.D., and Wiegand, S.J. (1999). Ves-
sel cooption, regression, and growth in tumors mediated by angio-
poietins and VEGF. Science 284, 1994–1998.
et al. (2001). Signalling via vascular endothelial growth factor recep-
tor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO
J. 20, 1223–1231.
Wiegand, S.J., Boland, P., and Yancopoulos, G.D. (2000). Coopera-
tive roles for the angiopoietins and vascular endothelial growth fac-
tor in ovarian angiogenesis. In Ovulation: Evolving Scientific and
Clinical Concepts, E.Y. Adashi, ed. (New York: Springer-Verlag), pp.
Wigle, J.T., and Oliver, G. (1999). Prox1 function is required for the
development of the murine lymphatic system. Cell 98, 769–778.
Yancopoulos, G.D., Davis, S., Gale, N.W., Rudge, J.S., Wiegand,
S.W., and Holash, J. (2000). Vascular-specific growth factors and
blood vessel formation. Nature 407, 242–248.
Zagzag, D., Hooper, A., Friedlander, D.R., Chan, W., Holash, J.,
Wiegand, S.J., Yancopoulos, G.D., and Grumet, M. (1999). In situ
expression of angiopoietins in astrocytomas identifies angiopoietin-2
Ito, M., and Yoshioka, M. (1999). Regression of the hyaloid vessels
and pupillary membrane of the mouse. Anat. Embryol. (Berl.) 200,
Jeltsch, M., Kaipainen, A., Joukov, V., Meng, X., Lakso, M., Rauvala,
Karkkainen, M.J., Ferrell, R.E., Lawrence, E.C., Kimak, M.A., Levin-
son, K.L., McTigue, M.A., Alitalo, K., and Finegold, D.N. (2000). Mis-
sense mutationsinterfere withVEGFR-3 signallingin primarylymph-
oedema. Nat. Genet. 25, 153–159.
Kukk, E., Lymboussaki, A., Taira, S., Kaipainen, A., Jeltsch, M., Jou-
kov, V., and Alitalo, K. (1996). VEGF-C receptor binding and pattern
of expression with VEGFR-3 suggests a role in lymphatic vascular
development. Development 122, 3829–3837.
Maisonpierre, P.C., Goldfarb, M., Yancopoulos, G.D., and Gao, G.
(1993). Distinct rat genes with related profiles of expression define
a TIE receptor tyrosine kinase family. Oncogene 8, 1631–1637.
Maisonpierre, P.C., Suri, C., Jones, P.F., Bartunkova, S., Wiegand,
dopoulos, N., et al. (1997). Angiopoietin-2, a natural antagonist for
Tie2 that disrupts in vivo angiogenesis. Science 277, 55–60.
Makinen, T., Jussila, L., Veikkola, T., Karpanen, T., Kettunen, M.I.,
Pulkkanen, K.J., Kauppinen, R., Jackson, D.G., Kubo, H., Nishikawa,
S., et al. (2001). Inhibition of lymphangiogenesis with resulting
receptor-3. Nat. Med. 7, 199–205.
expressing soluble VEGF
Mochizuki, Y., Nakamura, T., Kanetake, H., and Kanda, S. (2002).
Angiopoietin 2 stimulates migration and tube-like structure forma-
tion of murine brain capillary endothelial cells through c-Fes and
c-Fyn. J. Cell Sci. 115, 175–183.
Pierce, E.A., Avery, R.L., Foley, E.D., Aiello, L.P., and Smith, L.E.
tor expression in a mouse model of retinal neovascularization. Proc.
Natl. Acad. Sci. USA 92, 905–909.
Pierce, E.A.,Foley, E.D.,and Smith, L.E.(1996). Regulationof vascu-
lar endothelial growth factor by oxygen in a model of retinopathy
of prematurity. Arch. Ophthalmol. 114, 1219–1228.
Y., Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, W., and
Qin, Y. (1995). Distinct roles of the receptor tyrosine kinases Tie-1
and Tie-2 in blood vessel formation. Nature 376, 70–74.
Stone, J., Chan-Ling, T., Pe’er, J., Itin, A., Gnessin, H., and Keshet,
E. (1996). Roles of vascular endothelial growth factor and astrocyte
degeneration in the genesis of retinopathy of prematurity. Invest.
Ophthalmol. Vis. Sci. 37, 290–299.
Stone, J., Itin, A., Alon, T., Pe’er, J., Gnessin, H., Chan-Ling, T., and
Keshet, E. (1995). Development of retinal vasculature is mediated
by hypoxia-induced vascular endothelial growth factor (VEGF) ex-
pression by neuroglia. J. Neurosci. 15, 4738–4747.
Stratmann, A., Risau, W., and Plate, K.H. (1998). Cell type-specific
expression of Angiopoietin-1 and Angiopoietin-2 suggests a role in
glioblastoma angiogenesis. Am. J. Pathol. 153, 1459–1466.
Suri, C., Jones, P.F., Patan, S., Bartunkova, S., Maisonpierre, P.C.,
Davis, S., Sato, T.N., and Yancopoulos, G.D. (1996). Requisite role
of Angiopoietin-1, a ligand for the Tie2 receptor, during embryonic
angiogenesis. Cell 87, 1171–1180.
Teichert-Kuliszewska, K., Maisonpierre, P.C., Jones, N., Campbell,
A.I., Master, Z., Bendeck, M.P., Alitalo, K., Dumont, D.J., Yanco-
poulos, G.D., and Stewart, D.J. (2001). Biological action of angio-
poietin-2 in a fibrin matrix model of angiogenesis is associated with
activation of Tie2. Cardiovasc. Res. 49, 659–670.
Veikkola, T., Jussila, L., Makinen, T., Karpanen, T., Jeltsch, M., Pe-
trova, T.V., Kubo, H., Thurston, G., McDonald, D.M., Achen, M.G.,