Phosphatidylinositol 3-kinase signaling mediates
angiogenesis and expression of vascular endothelial
growth factor in endothelial cells
Bing-Hua Jiang, Jenny Z. Zheng, Masahiro Aoki, and Peter K. Vogt*
The Scripps Research Institute, Department of Molecular and Experimental Medicine, BCC239, 10550 North Torrey Pines Road, La Jolla, CA 92037
Contributed by Peter K. Vogt, December 21, 1999
Phosphatidylinositol 3-kinase (PI 3-kinase) is a signaling molecule
that controls numerous cellular properties and activities. The on-
cogene v-p3k is a homolog of the gene coding for the catalytic
subunit of PI 3-kinase, p110?. P3k induces transformation of cells
in culture, formation of hemangiosarcomas in young chickens, and
myogenic differentiation in myoblasts. Here, we describe a role of
PI 3-kinase in angiogenesis. Overexpression of the v-P3k protein or
of cellular PI 3-kinase equipped with a myristylation signal, Myr-
P3k, can induce angiogenesis in the chorioallantoic membrane
(CAM) of the chicken embryo. This process is characterized by
extensive sprouting of new blood vessels and enlargement of
PI 3-kinase target Akt, Myr-Akt, also induces angiogenesis. Over-
expression of the tumor suppressor PTEN or of dominant-negative
constructs of PI 3-kinase inhibits angiogenesis in the yolk sac of
chicken embryos, suggesting that PI 3-kinase and Akt signaling is
required for normal embryonal angiogenesis. The levels of mRNA
for vascular endothelial growth factor (VEGF) are elevated in cells
expressing activated PI 3-kinase or Myr-Akt. VEGF mRNA levels are
also increased by insulin treatment through the PI 3-kinase-depen-
dent pathway. VEGF mRNA levels are decreased in cells treated
with the PI 3-kinase inhibitor LY294002 and restored by overex-
pression of v-P3k or Myr-Akt. Overexpression of VEGF by the RCAS
vector induces angiogenesis in chicken embryos. These results
suggest that PI 3-kinase plays an important role in angiogenesis
and regulates VEGF expression.
Akt ? tumor suppressor ? transdominant negative mutant
3-kinase catalyzes the phosphorylation of inositol phospholipids
at the 3 position to generate phosphatidylinositol 3,4,5-
trisphosphate and phosphatidylinositol 3,4-bisphosphate. PI
3-kinase has been implicated in a number of cellular functions,
including cell adhesion, vesicular trafficking, protein synthesis,
and cell survival (1). An oncogenic form of PI 3-kinase has been
isolated from an avian retrovirus, ASV16, and termed v-P3k (2).
The oncogene v-p3k codes for a constitutively active form of the
catalytic subunit p110? of PI 3-kinase (2). Expression of the
v-P3k protein induces oncogenic transformation of chicken
ens, and formation of myotubes and myogenic differentiation in
chicken embryo myoblast cultures (2, 3).
The serine-threonine kinase Akt is a downstream target of PI
3-kinase. Akt is regulated by binding of its pleckstrin homology
domain to the lipid products of PI 3-kinase and by phosphory-
lation at Thr-308 and Ser-473 residues by two phosphoinositide-
dependent protein kinases, PDK1 and PDK2 (4, 5). Akt controls
cell survival, glycogen metabolism, cellular transformation, and
myogenic differentiation (6–10).
PI 3-kinase is activated by a variety of growth factors binding
to their receptors. Several of these growth factors [e.g., fibroblast
growth factors, epidermal growth factor, vascular endothelial
growth factor (VEGF), hepatocyte growth factor, and IL-8] are
hosphatidylinositol 3-kinase (PI 3-kinase) is activated by
insulin, by various growth factors, and by cytokines (1). PI
known to induce angiogenesis. Angiogenesis is required for the
progression of normal physiological events, such as embryonic
development, as well as for pathogenic processes, e.g., tumori-
genesis. In this report, we show that constitutively active PI
3-kinase and Akt induce angiogenesis. Inhibition of PI 3-kinase
signaling interferes with angiogenesis. PI 3-kinase signaling also
mediates VEGF expression in endothelial cells.
Materials and Methods
Plasmid Construction and Retrovirus Preparation. The plasmid con-
structs used are v-P3k, p85?iSH2, Myr-Akt, and quail VEGF
expressed by the avian retrovirus vector RCAS as previously
described (2, 3, 10, 11). PTEN (12, 13) and Myr-P3k, c-P3k fused
with the amino-terminal sequences of c-Src, were subcloned into
an adaptor vector pBSFI and then inserted into a modified avian
retrovirus vector RCAS.Sfi (10). Chicken embryo fibroblasts
(CEF) were plated at 2 ? 105cells?well in six-well plates the day
before the transfection, and transfected with 2 ?g of plasmid
DNA by using the Lipofectamine reagent (Life Technologies,
Gaithersburg, MD). After transfection, the cells were passaged
for 2 wk to ensure that the actively replicating retroviral vector
spreads through the culture. Infectious virus was harvested from
the cell culture medium and concentrated by centrifugation for
2 h at 23,000 rpm in a Beckman SW28 rotor at 4°C. The virus
pellet was resuspended in Ham’s F10 medium and stored at
Angiogenesis Assay in Chicken Embryos. Fertilized chicken eggs
(SPAFAS, Preston, CT) were incubated at 37°C with 70%
humidity for 9 days. An artificial air sac was created over a region
containing small blood vessels in the chicken chorioallantoic
membrane (CAM) as described (14). A small window was cut in
the shell over the artificial air sac. The CAM was infected with
15 ?l of RCAS, RCAS-v-P3k, or RCAS-Myr-Akt viruses con-
taining about 1–7 ? 108infectious units per ml. Angiogenesis
was monitored by photography 5, 7, and 9 days after infection.
The relative angiogenesis index was determined by measuring
the percentage of a unit area taken up by blood vessels (15) on
days 5 and 7 after infection by using the KS300 imaging system
Immunohistologic Analysis. CAM fragments were removed from
embryos 5 or 7 days after infection, and embedded in Tissue-Tek
O.C.T. compound (Sakura Finetek, Torrance, CA). The CAMs
Abbreviations: PI 3-kinase, phosphatidylinositol 3-kinase; CEF, chicken embryo fibroblasts;
CAM, chicken chorioallantoic membrane; CAME, chicken chorioallantoic membrane-
tensin homolog deleted on chromosome 10.
*To whom reprint requests should be addressed. E-mail: email@example.com.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073?pnas.040560897.
Article and publication date are at www.pnas.org?cgi?doi?10.1073?pnas.040560897
February 15, 2000 ?
vol. 97 ?
no. 4 ?
were snap frozen, and 6-?m cryostat sections were cut and fixed
in 3.7% formaldehyde in PBS. The CAM sections were blocked
for 1 h with 2% goat serum in PBS and incubated for 1 h with
a 1:50 dilution of rabbit anti-factor VIII antibodies (BioGenex
Laboratories, San Ramon, CA). After three 10-min washes with
PBS-0.2% Tween buffer, the sections were incubated with a
1:100 dilution of FITC-conjugated goat anti-rabbit IgG (Sigma)
for 1 h. After washes as above, the sections were mounted in and
examined with a fluorescence microscope (Zeiss).
Northern Blot Analysis. CEF and chicken endothelial cells pre-
pared from the CAM (CAME) were infected with virus express-
ing RCAS alone, v-P3k, Myr-P3k, or Myr-Akt. CEF were
cultured in cloning medium consisting of 83% Ham’s F-10, 10%
calf serum, 4.5% chicken serum, 1% of ?100 vitamin solution,
1% of 1.8 mM folic acid, and 0.5% DMSO. CAME cells were
cultured in cloning medium supplemented with 1% of endothe-
lial cell growth supplement (Upstate Biotechnology, Lake
reagent (Tel-Test, Friendswood, TX). Poly(A)?RNA was iso-
lated from total RNA by using Oligotex suspension (Qiagen,
Valencia, CA). Aliquots of 0.5 ?g poly(A)?RNA were frac-
tionated by formaldehyde gel electrophoresis and transferred to
a nylon membrane (Schleicher & Schuell). The blots were
hybridized with probes of quail VEGF cDNA (11) or actin
cDNA, and analyzed by autoradiography.
Results and Discussion
We used the oncoprotein v-P3k to investigate possible effects of
PI 3-kinase on angiogenesis in the chicken CAM. Expression of
CAM of chicken embryos. (A) Angiogenesis was assayed in 9-day-old chicken
embryos with 15 ?l of RCAS, RCAS-v-P3k, or RCAS-Myr-Akt viruses containing
about 1–7 ? 108infectious units per ml. The inocula were placed on the CAM
in a region devoid of blood vessels (15, 18). Angiogenesis was monitored by
photography 5, 7, and 9 days after infection. The experiments were repeated
at least four times with four to seven embryos for each virus, and represen-
tative portion on infected CAMs were photographed. The 5-day (5d) and
7-day (7d) photos were taken with ?6.5 magnification. The 9-day (9d) images
were taken with ?3 magnification. (B) The relative angiogenesis index was
determined by measuring the percentage of a unit area taken up by blood
vessels (15) on day 5 (5d) and 7 (7d) using the KS300 imaging system (Zeiss).
Data are expressed as mean from replicate experiments and normalized to
results obtained with CAMs infected by RCAS alone (?1).
Constitutively active PI 3-kinase and Akt induce angiogenesis in the
abnormally large capillaries and proliferation of endothelial cells. The CAMs
infected with virus as above were snap frozen, and 6-?m cryostat sections of
CAMs were fixed in 3.7% formaldehyde and stained by immunofluorescence
with factor VIII antibodies (BioGenex). (A) Representative fields showing
CAMs 7 days after infection by RCAS, RCAS-v-P3k, and RCAS-Myr-Akt virus
(?40 objective). (Upper) Immunofluorescence with factor VIII antibodies.
(Lower) Phase-contrast. (B) Representative fields were stained with factor VIII
antibodies, showing increased numbers of endothelial cells that form large
luminal structures 9 days after infection.
Constitutively active forms of PI 3-kinase and Akt induce multiple
www.pnas.orgJiang et al.
the proteins was mediated by the avian retroviral vector RCAS,
which produces infectious progeny virus that can spread in the
CAM. CAMs of 9-day chicken embryos (SPAFAS) were in-
fected with approximately 1.5–10.5 ? 106infectious units of
RCAS virus expressing v-P3k (2, 16, 17). The empty RCAS
retroviral vector served as control. High levels of v-P3k protein
were detected in the CAMs 3 days after infection with RCAS-
v-P3k. On day 5, an increase in the number and size of blood
vessels was evident in the v-P3k-expressing CAMs (Fig. 1A).
Sprouting of new vessels and enlargement of preexisting vessels
were observed. The expansion of the vasculature continued
rapidly, and, by day 7, small hemangiosarcomas could be de-
tected. On day 9, these had fused into one or a few large tumors
on each v-P3k-expressing CAM. In contrast, the CAMs of
control embryos infected with vector RCAS virus showed a
mature vascular system without excessive sprouting and branch-
ing of blood vessels. The effect of v-P3k on CAMs was quantified
by determining the relative angiogenesis index (15, 18), which is
based on the number of blood vessels per unit area on days 5 and
7 after infection, normalized to that of CAMs infected with the
RCAS vector. Expression of v-P3k increased the angiogenesis
index 3- to 5-fold relative to vector alone (Fig. 1B). Another
constitutively active form of PI 3-kinase, consisting of avian
cellular p110? with an amino-terminal myristylation signal,
Myr-P3k, also stimulated angiogenesis and induced hemangio-
sarcomas when expressed by RCAS in the CAM.
A downstream target of PI 3-kinase is the serine-threonine
protein kinase Akt, which serves as an essential component for
the transmission of anti-apoptotic (6–8), myogenic, and tumor-
igenic signals from PI 3-kinase (9, 10). To examine the possible
role of Akt in angiogenesis, CAMs from 9-day-old chicken
embryos were infected with 1.5–10.5 ? 106infectious units of
RCAS virus expressing Myr-Akt, a constitutively active form of
Akt generated by fusing a myristylation signal to the amino
terminus of the mouse cellular Akt. The development of neo-
vascularization and enlargement of existing vessels in these
CAMs were equal to or exceeded that seen with v-P3k or
Myr-P3k (Fig. 1).
Endothelial cells of blood vessels express factor VIII of the
blood clotting cascade and Flk-1, the receptor of VEGF. These
two proteins served as endothelial cell-specific markers (19, 20).
Their expression in CAMs was examined in cryostat sections
stained with fluorescent antibodies directed against factor VIII
or Flk-1. In control experiments, it was established that these
antibodies stain chicken endothelial cells prepared from normal
CAMs, but fail to stain CEF. CAMs expressing v-P3k or
alone 9 days after infection, and showed many abnormally large
capillaries and excessive proliferation of endothelial cells leading
to the formation of large luminal structures. Staining with factor
VIII antibodies, which marked the endothelial linings of the
blood vessels, was greatly increased (Fig. 2). Identical results
were obtained with antibodies against Flk-1.
PI 3-kinase is activated by growth factors through the inter-
action with the regulatory subunit p85. Mutants of p85 that fail
to bind to the catalytic subunit p110? function as dominant
negatives of PI 3-kinase signaling (3, 21). Another antagonist of
carrying p85?iSH2 or PTEN. Angiogenesis in yolk sac was monitored 9 days
after infection, and representative fields were photographed.
Overexpression of PI 3-kinase dominant negative constructs
alone, or RCAS expressing v-P3k, Myr-P3k, or Myr-Akt. CEF were cultured for
2 wk in cloning medium consisting of 83% Ham’s F-10, 10% calf serum, 4.5%
chicken serum, 1% of ?100 vitamin solution, 1% of 1.8 mM folic acid, and
1% of endothelial cell growth supplement (Upstate Biotechnology). mRNA
isolated from these cultures was analyzed in Northern blots. The blots were
hybridized with quail VEGF cDNA (11) or actin cDNA. (B) VEGF expression is
in CEF by the PI 3-kinase inhibitor, LY294002. Primary cultures of CAME and
CEF cells were switched to serum-free Ham’s F-10 medium for 24 h, and
remained untreated or were treated for 16 h with 150 nM insulin in the
presence of 40 ?M LY294002 or DMSO solvent. Northern blots were per-
formed as above. (C) v-P3k and Myr-Akt reverse the LY294002-induced inhi-
bition of VEGF expression. CAME cells were cultured in cloning medium
supplemented as described and infected with RCAS, RCAS-v-P3k, or RCAS-
Myr-Akt. Two weeks after infection, the cells were switched to serum-free
Ham’s F10 for 24 h and treated with 150 nM insulin in the presence of 40 ?M
LY294002 (LY, ?) or DMSO (LY, ?).
Jiang et al.
February 15, 2000 ?
vol. 97 ?
no. 4 ?
PI 3-kinase is PTEN, a phosphatase that removes the phosphate
at the 3 position of phosphatidylinositol-3,4,5-trisphosphate
(22–24). Introduction of either dominant-negative p85 or PTEN
into 4-day-old chicken embryos with the RCAS vector inhibited
numbers and sizes of blood vessels in yolk sac development (Fig.
3), presumably by interfering with endogenous PI 3-kinase and
Akt signaling. These results suggest that PI 3-kinase signaling is
required for normal embryonal angiogenesis.
PI 3-kinase and Akt probably stimulate angiogenesis through
the activation of one or several downstream mediators. A
candidate for this mediator function is VEGF, which specifically
stimulates endothelial cell mitogenesis. VEGF is induced by
v-Src and by activated Ras (25–28), both are known to activate
PI 3-kinase. We therefore expressed v-P3k, Myr-P3k, or Myr-
Northern blot analysis demonstrated that activated PI 3-kinase
and Akt both increased the levels of VEGF mRNA (Fig. 4A). To
test whether insulin can induce VEGF expression in a PI
3-kinase-dependent manner, CEF and CAME cells were
switched to serum-free Ham’s F10 medium for 24 h and then
stimulated with 150 nM insulin for 16 h in the presence or
absence of PI 3-kinase inhibitor LY294002. VEGF mRNA was
induced by insulin in both CAME and CEF cells. The inhibitor
LY294002 prevented VEGF expression specifically in CAME
cells but not in CEF (Fig. 3B). The reduction in VEGF expres-
sion by LY294002 was partially relieved by v-P3k and was
completely reversed by Myr-Akt (Fig. 4C). The effect of VEGF
overexpression was investigated by infecting CAMs with the
RCAS-VEGF construct (11). Seven days after inoculation,
extensive sprouting of blood vessels and expansion of preexisting
vessels was observed, but overexpression of VEGF failed to
induce fusion of blood vessels or hemangiosarcomas (Fig. 5).
Angiogenesis is critical in the development of tumors (29–33),
in ocular neovascularization (29, 34, 35), and in inflammation
(36, 37). The results described in this paper identify signals from
PI 3-kinase and from its target Akt as regulators of angiogenesis.
Activated PI 3-kinase and Akt are strong inducers of neovascu-
larization and endothelial cell proliferation. Embryonal angio-
genesis is interfered with by dominant-negative PI 3-kinase and
by the PI 3-kinase antagonist PTEN. RNA levels of VEGF are
elevated in cells expressing activated PI 3-kinase and Akt either
by enhanced transcription or increased RNA stability. Tran-
scription of a reporter construct containing promoter-enhancer
sequences of the human VEGF gene is stimulated by PI 3-kinase
and Akt (data not shown). The induction of VEGF by insulin is
inhibited by the PI 3-kinase inhibitor LY294002 in endothelial
cells, and this inhibition can be relieved or completely abolished
growth factor also requires PI 3-kinase (38). VEGF has been
shown to induce the activation of PI 3-kinase and Akt in human
umbilical vein endothelial cells (39, 40). Although VEGF treat-
ment of CAMs induced angiogenesis, it did not increase Akt
activity in CAME cell culture (data not shown), suggesting that
in this cell system there is no feedback regulatory loop that
includes VEGF and PI 3-kinase. PI 3-kinase also plays an
important role in embryonal angiogenesis mediated by VE-
cadherin (41) and by the receptor tyrosine kinase Tie2 (42).
Although VEGF is up-regulated by PI 3-kinase and Akt and
induces angiogenesis in the CAM, it may not be the only
angiogenesis-related target of PI 3-kinase signaling. Overexpres-
sion of VEGF with the RCAS vector does not induce heman-
giosarcomas; therefore, additional factor(s) must mediate the
oncogenic effect of PI 3-kinase and of Akt on endothelial cells.
An important open question concerns the downstream compo-
nents of the angiogenic PI 3-kinase?Akt signals, especially the
identity of the transcriptional regulators that affect VEGF
expression in response to PI 3-kinase and Akt signaling. Hy-
poxia-inducible factor 1 (43) and activator protein-1 (44) have
been implicated in the transcriptional activation of VEGF.
Experiments are in progress to define the PI 3-kinase-responsive
element in the VEGF promoter and identify the relevant
We thank W. K. Cavenee for PTEN, and W. Risau and I. Flamme for
quail VEGF expression plasmids. This work was supported by U.S.
Public Health Service Grants CA 42564 and 78230 (to P.K.V.) and the
Sam and Rose Stein Endowment Fund. B.-H.J. is the recipient of
National Research Service Award postdoctoral fellowship CA 77892
from the National Cancer Institute.
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