Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability.
ABSTRACT Src kinase activity was found to protect endothelial cells from apoptosis during vascular endothelial growth factor (VEGF)-, but not basic fibroblast growth factor (bFGF)-, mediated angiogenesis in chick embryos and mice. In fact, retroviral targeting of kinase-deleted Src to tumor-associated blood vessels suppressed angiogenesis and the growth of a VEGF-producing tumor. Although mice lacking individual Src family kinases (SFKs) showed normal angiogenesis, mice deficient in pp60c-src or pp62c-yes showed no VEGF-induced vascular permeability (VP), yet fyn-/- mice displayed normal VP. In contrast, inflammation-mediated VP appeared normal in Src-deficient mice. Therefore, VEGF-, but not bFGF-, mediated angiogenesis requires SFK activity in general, whereas the VP activity of VEGF specifically depends on the SFKs, Src, or Yes.
- SourceAvailable from: Paul L Stein[show abstract] [hide abstract]
ABSTRACT: Three members of the Src family of tyrosine kinases, src, fyn, and yes, are broadly expressed throughout mouse development. Mutations in the c-src and fyn genes were shown previously to lead to restricted nonoverlapping phenotypes only in a subset of cells in which these kinases are expressed. In this work we show that a mutation in the yes gene does not lead to an overt phenotype. Except for brain, the level or distribution of related kinases is not altered in major tissues. To gain further insight into the possibility that these kinases compensate for each other, animals deficient in multiple src-kinases were generated. Whereas most of the src/fyn or src/yes double mutants die perinatally, a substantial proportion of fyn/yes double mutants are viable but undergo degenerative renal changes leading to diffuse segmental glomerulosclerosis. Taken together, these data are consistent with the hypothesis that, at least in some cells, these kinases are able to compensate for the loss of the other related kinases.Genes & Development 10/1994; 8(17):1999-2007. · 12.44 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Angiogenesis depends on growth factors and vascular cell adhesion events. Integrins and growth factors are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic fibroblast growth factor (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least 20 h. The initial wave of ERK activity (5-120 min) was refractory to integrin antagonists, whereas the sustained activity (4-20 h) depended on integrin alphavbeta3, but not beta1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained alphavbeta3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked growth factor induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a growth factor receptor and integrin alphavbeta3.The Journal of Cell Biology 04/1998; 140(5):1255-63. · 10.82 Impact Factor
- The Journal of Physiology 11/1952; 118(2):228-57. · 4.38 Impact Factor
Molecular Cell, Vol. 4, 915±924, December, 1999, Copyright 1999 by Cell Press
Selective Requirement for Src Kinases
during VEGF-Induced Angiogenesis
and Vascular Permeability
forms of Src or intact C-terminal Src kinase (Csk) to
disrupt endogenous Src activity within the chick chorio-
allantoic membrane (CAM) or mouse skin to directly
evaluatethegeneralroleofSrc kinases during angiogen-
esis. Evidence is provided here that Src kinase is re-
quired forVEGF-, butnotbFGF-, mediated angiogenesis
in both the chick embryo and the mouse. In fact, Src
kinase activity was found to be required for endothelial
cellsurvival during VEGF-mediated angiogenesis. While
VEGF is an endothelial cell mitogen (Ferrara and Davis-
Smyth, 1997), it was originally described forits vascular
permeability (VP) activity (Senger et al., 1983; Connolly
et al., 1989). In fact, VEGF is unique in this regard, as
other growth factors such as bFGF can induce neovas-
cularization but do not induce vascular permeability
(Connolly et al., 1989; Murohara et al., 1998). Ananalysis
of mice deficient in specific SFKs demonstrates no de-
crease in VEGF-dependent neovascularization but a
complete ablation of its VP activity in src?/?or yes?/?
mice, while fyn?/?mice show no such defect. While mice
lacking Src show no VP response to VEGF, they do show
a VP response to an inflammatory mediator. Therefore,
multiple SFKs can serve as key signaling intermediates
involved in VEGF-induced vascular proliferation, while
the VP activity of this growth factor depends on Src or
Yes in particular.
Brian P. Eliceiri,*Robert Paul,*
Pamela L. Schwartzberg,²J ohn D. Hood,*
J ie Leng,*and David A. Cheresh*³
*Departments of Immunology and Vascular Biology
The Scripps Research Institute
10550 North Torrey Pines Road
La J olla, California 92037
²The National Human Genome Research Institute
National Institutes of Health
Bethesda, Maryland 20892
Src kinase activity was found to protect endothelial
cells from apoptosis during vascular endothelial growth
factor (VEGF)±, but not basic fibroblast growth factor
(bFGF)±, mediated angiogenesis in chick embryos and
mice. In fact, retroviral targeting of kinase-deleted Src
to tumor-associated blood vessels suppressed angio-
genesis and the growth of a VEGF-producing tumor.
Although mice lacking individual Src family kinases
(SFKs) showed normal angiogenesis, mice deficient in
pp60c-srcorpp62c-yesshowed no VEGF-induced vascular
permeability (VP), yet fyn?/?mice displayed normal
VP. In contrast, inflammation-mediated VP appeared
normal in Src-deficient mice. Therefore, VEGF-, but
not bFGF-, mediated angiogenesis requires SFK activ-
ity in general, whereas the VP activity of VEGF specifi-
cally depends on the SFKs, Src, or Yes.
Src Activity Is Required for VEGF-, but Not bFGF-,
To establishwhetherendogenous Src activity was asso-
ciated with growth factor±mediated angiogenesis, filter
disks saturated with either bFGF or VEGF were placed
ontheCAMof10-day-old chick embryos.This treatment
is known to promote a robust angiogenic response as
measured after 48±72 hr (Brooks et al., 1994a). Lysates
of these CAMs were evaluated forSrc activity by immu-
noprecipitating Src and measuring its ability to phos-
phorylate a GST±focaladhesionkinase (FAK)fusionpro-
tein in an in vitro kinase assay. At least a 2-fold increase
inendogenous Src activity was detected intheselysates
120 min after the addition of either bFGF or VEGF to
the CAM tissue (Figure 1A). Importantly, we observed a
similar increase in Src activity 15 min after the addition
of either growth factor (data not shown). To determine
whether Src activity was required for angiogenesis,
CAMs stimulated with either bFGF or VEGF were in-
fected with an avian-specific retroviral vector (RCAS)
containing a truncationmutantofSrc lacking its C-termi-
nal kinase domain (Src 251) (Kaplan et al., 1994). This
Src 251and similartruncationmutants havebeenshown
to function as a dominant negative of multiple SFKs,
thereby blocking signaling events downstream of growth
factor receptors (Broome and Hunter, 1996; P. L. S.,
unpublished data). The RCAS retrovirus, when applied
to CAM tissues, infects fibroblasts and endothelial cells
proximal to the filter disk as determined by infecting
CAMs with an RCAS±GFP vector and examination by
SFKs are important signaling molecules that respond to
a wide range of stimuli including growth factors (Twam-
ley-Stein et al., 1993; Broome and Hunter, 1996) and
adhesion proteins in the extracellular matrix (Kaplan et
al., 1994; Schwartz et al., 1995; Thomas and Brugge,
1997; Klinghoffer et al., 1999). Once activated, SFKs
affect a wide range of downstream signaling events in-
cluding the activation of MAP kinases (Courtneidge et
al., 1993). While in vitro studies have elucidated a role
forSrc incellularfunction, due to mechanisms ofredun-
dancies and compensation, mice lacking a single SFK
(Soriano et al., 1991; Stein et al., 1994) have provided
limited insight into the biological function of this impor-
tant family of nonreceptor tyrosine kinases.
Previous studies have implicated SFKs in vascular
cellproliferation. Forexample, v-Src, anoncogenic vari-
ant of Src, is known to promote hemangioma formation
in chicks (Stoker et al., 1990), suggesting that under
normalcircumstances, c-Src orotherSFKs mayregulate
the growth of blood vessels. To initially address this
issue, we used avian- ormurine-targeted retroviraldeliv-
ery systems to express mutationally active or inactive
³To whomcorrespondence should be addressed (e-mail: cheresh@
Figure 1. Activation of Endogenous Src Ki-
nase Activity by bFGF and VEGF and the Ef-
fect of Kinase-Deleted Src on Angiogenesis
(A) Tissue extracts of 10-day-old chick CAMs
were exposed to filter paper disks saturated
withbFGF orVEGF (2 ?g/ml)for2 hr.Endoge-
nous Src was immunoprecipitated from equiv-
alent amounts of total protein and subjected
to an in vitro immune complex kinase assays
witha FAK±GST fusionproteinas a substrate,
electrophoresed, and transferred to nitrocel-
lulose. The relative fold increase in Src activ-
ity is indicated in italics. The above kinase
assay blot was probed with an anti-Src anti-
body as a loading control for equivalent Src
and IgG content.
(B) Chick CAMs (9 day) were exposed to filter
paperdisks saturated withRCAS±Src 251 (ki-
nase deleted) or RCAS±GFP containing re-
troviruses or buffer for 20 hr and then incu-
bated in the presence or absence of bFGF or
VEGF for an additional 72 hr. Tissue extracts
of these CAMs were examined for endoge-
nous Src activity by in vitro immune complex
kinase assay as described above using FAK±
GST as a substrate.
(C) The level of angiogenesis was quantified
in embryos incubated with RCAS±Src251 or
RCAS±GFP followed by stimulation with ei-
ther bFGF or VEGF as described above.
Blood vessels were enumerated by counting
blood vessel branch points in a double
blinded manner. Each bar represents the
mean ? SEM of three replicates.
(D)Micrographs ofrepresentativeCAMs were
taken with an Olympus stereomicroscope.
Scale bar, 350 ?m.
confocal microscopy (data not shown). Delivery of this
kinase-deleted Src completely disrupted endogenous
Src kinase activity in these tissues induced with either
growth factor (Figure 1B).
To examine the role of Src in angiogenesis, CAMs
stimulated with either bFGF or VEGF were infected with
the Src 251±containing retrovirus. As shown in Figure
1C, angiogenesis, as measured 72 hr after stimulation
withVEGF, was suppressed by delivery ofSrc 251; how-
ever, to our surprise, bFGF-induced angiogenesis was
not affected. Importantly, equivalent levels ofviralinfec-
tionweredetected inVEGF- andbFGF-stimulated CAMs
as measured by epifluorescence and immunoblotanaly-
sis of GFP and Src 251, respectively (data not shown).
The inhibitionofVEGF-induced angiogenesis by kinase-
deleted Src was likely due to a direct effect on endothe-
lialcells, since VEGF is a knownendothelialcell-specific
mitogen. In addition, the failure of Src 251 to disrupt
bFGF-induced angiogenesis indicates that the effects
on VEGF-mediated angiogenesis are not due to general
toxicity. Together, these results demonstrate that, while
both bFGF and VEGF can activate Src kinase in these
tissues, only VEGF-induced blood vessel formation re-
quired this activity. These findings support the recent
reports thatVEGF and bFGF stimulate distinctpathways
of angiogenesis (Friedlander et al., 1995; Ziche et al.,
Suppression of Human Tumor Growth by Targeting
the Tumor Vascular Compartment with
Retroviral Delivery of Src 251
Tumorgrowthdepends onangiogenesis (Weidneret al.,
1991; Folkman and Shing, 1992; Brooks et al., 1994b).
In fact, recent reports suggest that tumorgrowth is sus-
ceptible to the antiangiogenic effects of VEGF receptor
antagonists (Kim et al., 1993). Therefore, experiments
were designed to determine whether suppression of an-
giogenesis by delivery of kinase-deleted Src 251 would
influence the growth of a human medulloblastoma
(DAOY), a highly angiogenic tumor known to produce
VEGF and very little bFGF (data not shown). This human
tumorreadily grows onthe CAM and produces anactive
angiogenic response (Figure 2), allowing us to selec-
tively target the tumor vasculature by using the avian-
specific RCAS retrovirus, without infecting the human
medulloblastoma cells. Delivery ofRCAS containing Src
251 to preestablished medulloblastomas resulted in a
selective expressionofthevirus inthe tumor-associated
Src Requirement for Angiogenesis and Permeability
fewer in number compared to the tumor vessels in
control animals (Figure 2C). The fact that RCAS-GFP-
infected tumors showed GFP localization only in the
tumor vasculature suggests that the antitumor effects
observed with retrovirally delivered Src 251 were due
to its targeting and antiangiogenic properties.
Src Requirement for Endothelial Cell Survival
during VEGF-, but Not bFGF-,
Recent evidence suggests that growth factor receptors
(Choi and Ballermann, 1995; Satake et al., 1998) and
integrins (Meredith et al., 1993; Brooks et al., 1994a)
promote survival of angiogenic endothelial cells. The
fact that both growth factors and adhesion receptors
also regulate Src activity prompted us to examine the
role of Src in endothelial cell survival during angiogen-
esis. Furthermore, the Src 251 mutant has been found
to induce apoptosis in selective cell types during bone
development (P. L. S., L. Xing, and B. Boyce, unpub-
lished data). CAMs stimulated with eitherbFGF orVEGF
were infected with retrovirus containing Src 251, and
cryostat sections of these tissues were examined for
the presence of apoptotic cells. As shown in Figure 3A,
delivery of Src 251 promoted extensive TUNEL staining
among the factor VIII±related antigen (von Willebrand
factor [vWf]) positive blood vessels in VEGF-, but not
bFGF-, stimulated CAMS.Infact, minimalapoptosis was
observed among other cell types in these CAMs (Figure
3), suggesting an endothelial cell-specific requirement
forSrc kinase activity forcell survival in VEGF-activated
blood vessels. In a second series of experiments, retro-
virus-infectedCAMs stimulatedwithVEGF orbFGF were
subjected to limited collagenase digestion to prepare a
single cell suspension. These CAM-derived cells were
shown to contain approximately 20%±50% endothelial
cells (vWf positive) (Figures 3C and 3D) and analyzed
for apoptosis by flow cytometric detection of annexin
V±positive cells, an early apoptosis marker. As shown
in Figure 3B, cells derived from VEGF-stimulated CAMs
infected with Src 251 had significantly increased an-
nexin V staining relative to cells from mock RCAS-GFP-
infected CAMs treated with VEGF. In contrast, cells de-
rived from mock-infected CAMs or those infected with
RCAS±Src 251 and stimulated with bFGF exhibited little
or no annexin V staining (data not shown). In addition,
no annexinV staining was detected among cells derived
fromnonstimulated orbFGF-stimulated CAMs (data not
shown). These data demonstrate that Src kinase activity
is selectively required forendothelial cell survival during
VEGF, butnotbFGF-mediated angiogenesis inthe CAM.
Figure 2. Retroviral Delivery of RCAS±Src 251 to Human Tumors
Growing on the Chick CAM Reverses Tumor Growth
(A) Human DAOY medulloblastomas, which express VEGF, were
grownonthe CAM ofchick embryos as described inthe Experimen-
tal Procedures. Retrovirus containing RCAS±GFP or RCAS±Src 251
was topically applied to preestablished tumors of greater than 50
mg. A representative micrograph of a medulloblastoma tumor frag-
ment infected with RCAS±GFP expressing GFP reveals exclusive
expression in the tumor blood vessels (arrowhead) as detected by
optical sectioning with a Bio-Rad 1024 laser confocal scanning mi-
croscope. Scale bar, 500 ?m.
(B) Tumors treated as above were allowed to grow for 3 or 6 days,
after which they were resected and wet weights were determined.
Data are expressed as the mean change in tumor weight (from the
50 mg tumor starting weight) ? SEM of two replicates. RCAS±Src
251 had a significant impact on tumor growth after 3 days (*p ?
0.002) and 6 days (**p ? 0.05).
(C) Representative stereomicrographs of medulloblastoma tumors
surgically removed from the embryos were taken with an Olympus
stereomicroscope (scale bar, 350 ?m). (Lowerpanel)A highmagnifi-
cation micrograph of each tumor showing the vasculature in detail
in RCAS±Src 251±treated tumors.
Selective Requirement for Src Kinase Activity
in a Subcutaneous Murine Model
To further analyze the role of Src in angiogenesis, a
murine model was employed. In this case, angiogenesis
was induced by subcutaneous injection of growth fac-
tor±depleted Matrigel supplemented with either bFGF
(400 ng/ml) or VEGF (400 ng/ml) in athymic wehi (nu/nu)
adult mice and analyzed after 5 days (Passaniti et al.,
1992). Angiogenesis was quantitated by removing and
extracting the angiogenic tissue and thensubjecting the
blood vessels (Figure 2A), which led to a complete sup-
pression of tumor growth (Figure 2B). Importantly, the
tumor-associated blood vessels in animals treated with
virus containing Src 251 were severely disrupted and
Figure 3. Apoptosis in VEGF-Stimulated Blood Vessels Expressing Src 251
(A) Immunolocalization of factor VIII±related antigen (von Willlebrand factor), apoptag immunostaining of apoptotic cells, and nuclear staining
with DAPI in cryosections of CAMs expressing RCAS±Src 251 or RCAS±GFP, after stimulation with bFGF or VEGF as described in Figure 1.
The merge represents an overlay of the factor VIII staining and apoptag staining. The fluorescence from the GFP was not preserved in the
fixation protocol used for the indirect immunofluorescence in these experiments. These micrographs were representative of blood vessel
staining in duplicate samples. Scale bar, 50 ?m.
(B) Apoptotic cells were identified by annexin V staining of RCAS±Src 251±infected CAMS treated with VEGF and detected by flow cytometry.
Collagenase-dissociated cells isolated from RCAS±Src 251± (black) or RCAS-GFP- (mock, white) infected CAMs treated with VEGF, as
described in Figure 1, were incubated with annexin V. The fluorescence from the GFP was not detected in these assays, and the FACS profile
was similar to untreated controls. The flow cytometry data for each experiment was representative of at least three replicates.
(C) Anti-vWf staining was detected with a FITC-labeled secondary antibody used to identify endothelial cells by flow cytometry, and this was
compared to parallel collagenase-dissociated untreated CAM cells incubated without primary antibody.
(D) Immunolocalization of endogenous von Willebrand factor in collagenase-dissociated untreated permeabilized CAM cells (arrowhead)
replated on 3 ?g/ml collagen and detected with a fluorescent secondary antibody (bar, 10 ?m).
lysates to immunoblotting with a VEGF receptor anti-
body (flk-1) (Figure 4A) that is specific for endothelial
cells.As observed inthe chick, expressionofthe kinase-
deletedSrc 251cDNA blockedVEGF-inducedangiogen-
esis inthis murinemodelwhilehaving noeffectonbFGF-
induced angiogenesis (Figure 4B). To establish the role
of endogenous Src in this model, tissues were infected
with a retrovirus expressing Csk that inhibits endoge-
nous Src activity by phosphorylation of the C-terminal
regulatory site (Nada et al., 1991). Expression of Csk
blocked VEGF-, but not bFGF-, induced angiogenesis
(Figure 4), confirming a role forendogenous Src activity
in VEGF-mediated angiogenesis. Neovascularization of
these virus-infected VEGF-stimulated tissues was con-
firmed by directimmunofluorescencewitha FITC-conju-
gated anti-CD34 antibody (Figure 4) oran anti-flk-1 anti-
body (data not shown) and quantitated by enumerating
the number of positively stained CD34 blood vessels in
each cryosection (Figure 4C).
The Effect of Intradermal Expression of VEGF
in src?/?or src?/?Mice Ears
To extend the observations made in the chicken and
mouse angiogenesis models, a direct genetic approach
was employed to examine intradermal VEGF-induced
angiogenesis insrc?/?mice. We also considered the fact
that VEGF both initiates new blood vessel growth and
can promote vascular permeability (Senger et al., 1983;
Ferrara and Davis-Smyth, 1997). Intradermal injections
of adenovirus expressing a human VEGF cDNA were
performed in the ear of src?/?and src?/?, while control
?-galactosidase expressing adenovirus was injected
into the opposite ear of each mouse. VEGF-dependent
Src Requirement for Angiogenesis and Permeability
5A, which confirms the extent of the vascular leakage
in src?/?mice that is essentially absent in the src?/?
mice. The vascular leakage in these animals suggested
that the VP activity, which has been associated with
angiogenesis invivo(Dvorak etal.,1995),couldbeselec-
tively disrupted in pp60c-src-deficient mice.
VEGF Fails to Compromise the Blood±Brain
Barrier in Mice Lacking pp60c-src
The brain vasculature is characterized by a highly re-
strictive blood±brain barrier that prohibits small mole-
cules from extravasating into the surrounding brain tis-
sue. Tumor growth within the brain can compromise
this barrier due in part to the production of angiogenic
growth factors such as VEGF. Therefore, we examined
the nature of the blood±brain barrier in src?/?or src?/?
mice. In this case, VEGF or saline was stereotactically
injected into the right or left hemisphere of the brain,
respectively. All mice received systemic injections of
Evan's blue to monitor VP activity. As shown in Figure
5B, vascularleakageofblood was localized totheVEGF-
injected hemisphere insrc?/?mice, butthere a complete
absence of vascular leakage in src?/?mice. This was
also the case when examining the VP by measuring
the accumulation of Evan's blue dye as detected by
epifluorescence analysis of cryostat sections of these
brains (Figure 5C). Thus, VEGF compromises the blood±
brain barrier in a manner that depends on pp60c-src.
VEGF-Mediated VP, but Not Inflammation-
Associated VP, Depends on pp60c-src
To further analyze and quantitate the effect of VEGF as
a VP factor in src?/?or src?/?mice, we used the Miles
assay (Miles and Miles, 1952) to quantitatively measure
the vascular permeability in the skin of these animals.
VEGF was injected intradermally in src?/?or src?/?mice
that had received an intravenous systemic administra-
tion of Evan's blue dye. Within 15 min after injection of
VEGF, there was a 3-fold increase in VP in src?/?. How-
ever, insrc?/?mice, we observed no detectable VP activ-
ity (Figures 6A and 6B). Dye elution of the injected skin
patches was quantitated and compared with control
saline and bFGF (Figure 6B, left panel). bFGF or saline
controls injected adjacenttotheVEGF showednosignif-
icant increase in VP.
Vascular leakage/permeability is also known to occur
during inflammation, which allows for the accumulation
ofserum-associated adhesive proteinand inflammatory
cells in tissues. In fact, inflammatory mediators them-
selves directly promote vascularleakage. Therefore, we
tested one suchinflammatory mediator, allylisothiocya-
nate, also known as mustard oil (Inoue et al., 1997), in
src?/?or src?/?mice for its capacity to produce VP.
Unlike that observed in VEGF-stimulated src?/?animals,
ofthe inflammatory mediatorallylisothiocyanate (Figure
6B, right panel). Thus, we conclude that Src plays a
selective role in the VP activity induced with VEGF and
does not influence VP associated with the inflammatory
Figure 4. Retroviral Delivery of Src 251 and Csk in a Subcutaneous
Murine Angiogenesis Model
(A)Angiogenesis was inducedby asubcutaneous injectionofgrowth
factor-depleted Matrigel containing saline or VEGF (400 ng/ml) with
2 ? 106ecotropic packaging cells expressing GFP retrovirus in the
flank ofathymic wehi(nu/nu)mice and analyzed after5 days ofincuba-
tion. The neovascularization was quantitated by immunoblotting
with a VEGF receptor antibody (flk-1) that is specific for endothelial
(B) The effects of kinase-deleted Src 251, Csk, or GFP retrovirus on
VEGF- (400 ng/ml) or bFGF- (400 ng/ml) induced angiogenesis was
analyzed by immunoblotting the tissue lysates with an anti-flk-1
(C) The effect of the Src 251± and Csk-expressing retroviruses on
VEGF-induced neovascularization was quantified by enumerating
the number of CD34 positive vessels in tissue cross sections by
indirect immunofluorescence in triplicate random fields at 20? as
described in the Experimental Procedures.
new blood vessel growth in src?/?ears was first detect-
able within 48 hr, and neovascularization was analyzed
after 5 days (Figure 5A). There were identical viral ex-
pression levels in src?/?and src?/?as determined by
X-gal staining of ?-galactosidase-adenovirus injected
was no significant decrease in angiogenesis (data not
shown) as measured by counting branch points (p ?
0.05). However, the most apparent phenotype in these
animals was the complete blockade inthe vascularleak-
age compared to the VEGF-injected src?/?ears. Repre-
sentative ears injected with VEGF are shown in Figure
Figure 5. TheEffectofVEGF-InducedVascu-
lar Leakage in the Ears and Brains of src?/?
(A) Gene delivery of the human VEGF cDNA
inanadenovirus vectorwas injected intrader-
mally in the right ear of src?/?or src?/?mice,
and the neovascularization of the ears were
photographed after5 days ofexpression. Ad-
enovirus expressing ?-galactosidase was in-
jected into the left ears as a negative control.
Staining for ?-galactosidase in these ears
confirmed similar adenovirus expression in
each genetic background. Scale bar, 1 mm;
n ? 4.
(B) VEGF or saline was stereotactically in-
jected into the left or right frontal lobes, re-
spectively, of src?/?or src?/?. After injection
with Evan's blue and perfusion, the brains
were removed and photographed with a ste-
reoscope (6?, final magnification; arrow-
head, injection site).
(C) Cross sections of the above VEGF- or
saline-injected brains from src?/?or src?/?
mice were prepared and analyzed for VEGF-
induced VP by confocalmicroscopy to visual-
ize the fluorescence of the extravasated Ev-
an's blue (6?, finalmagnification; arrowhead,
VEGF-Mediated VP Activity Depends on Src
and Yes but Not Fyn
We next tested the specificity of the Src requirement
forVP by examining the VEGF-induced VP activity asso-
ciated with SFKs such as Fyn or Yes, which, like Src,
are known to be expressed in endothelial cells (Bull et
al., 1994; Kiefer et al., 1994). In fact, we confirmed that
these three SFKs were expressed equivalently in the
aortas of wild-type mice (data not shown). Like src?/?
mice, animals deficient in Yes were also defective in
VEGF-induced VP (Figure 6C). However, to oursurprise,
mice lacking Fynretained a highVP inresponse to VEGF
that was not significantly different from control animals
(Figure 6C). The disruption of VEGF-induced vascular
permeability in src?/?or yes?/?mice demonstrates that
thekinase activity ofspecific SFKs is essentialforVEGF-
mediated signaling event leading to VP activity but not
naling events required for the growth of new blood ves-
sels. In this report, evidence is provided that two angio-
genic growth factors, bFGF and VEGF, initiate signaling
pathways that can be distinguished based on their re-
quirement for Src kinase activity. Even though both
bFGF and VEGF led to increased Src activity in angio-
genic tissues,onlyVEGF-inducedangiogenesis depended
on it. This was based on studies where kinase-deleted
Src orCsk was retrovirally delivered to stimulated blood
vessels. The use ofintact Csk was importantas itblocks
the activity of endogenous Src rather than acting as a
dominant-negative mutant like Src 251. Src activity was
found to be required forthe survival of VEGF-stimulated
endothelial cells in vivo.
VEGF was originally described as a vascular perme-
ability factor secreted by tumor cells (Senger et al.,
1983). Using mice deficient for specific SFKs, we dem-
onstrated that pp60c-srcor pp62c-yesare essential for
VEGF-induced VP, while its angiogenic activity was not
significantly influenced in these animals. Moreover, ani-
mals deficient inFyn show no loss ofVP activity demon-
strating that only certain SFKs are required to regulate
VEGF-mediated VP activity. Importantly, all three of
While multiple growth factors and adhesion events can
promote angiogenesis, little is known regarding the sig-
Src Requirement for Angiogenesis and Permeability
other postnatal process. Interestingly, mice lacking the
combination of Src, Yes, and Fyn or the VEGF receptor
show embryonic lethality by day 9.5, a timeduring devel-
opment that is characterized by active vasculogenesis
(Fong et al., 1995; Shalaby et al., 1995). This, together
with the fact that mice lacking individual SFKs develop
normalappearing blood vessels, suggests thatcompen-
sation can take place among these SFKs. This is sup-
portedby ourobservationthatsuppressionofSrc kinase
activity ingeneralby Csk orSrc251 suppressed neovas-
cularization in mice or chick embryos in response to
VEGF while individual SFK knockouts develop normally.
Evidence provided in this study demonstrates that
VEGF and bFGF potentiate somewhat different biologi-
cal and biochemical effects during the early stages of
angiogenesis. There may be a physiologicalrationale for
the existence oftwo angiogenic pathways.Forexample,
blood vessels in various organs may differ with respect
to distinct ECM-associated adhesive proteins and/or
growth factors. Neovascularization in the retina has
been linked to VEGF expression (D'Amore, 1994; Miller
etal., 1994), while thatinduced during cutaneous wound
repair has been associated with bFGF (Takenaka et al.,
1997). This may allow endothelial cells to meet the spe-
cific needs of a given tissue depending on local require-
ments for nutrients, oxygen, or waste elimination. After
hypoxic injury, VEGF levels are known to rise immedi-
ately (reviewed in Ferrara and Davis-Smyth, 1997). Per-
haps this hypoxic response facilitates an immediate in-
creased oxygenation by providing local vascular leakage
prior to the actual formation of a new vascular network.
This would predict that adult pp60c-src- or pp62c-yes-defi-
cientmice may be less capable ofrestoring oxygenation
to damaged or hypoxic tissue. In fact, we noted that
stereotactic injectionofVEGF inthebraincould compro-
mise the blood±brain barrier in control animals. How-
ever,animals deficientinpp60c-srcshowed nobreakdown
of the blood±brain barrier.
VEGF is an angiogenic growth factor in many tumors.
In fact, an anti-VEGF antibody (Kim et al., 1993) that
blocks tumorgrowthinmice is being evaluated clinically
in patients with late-stage cancer. Given the strong as-
sociation between VEGF and tumor angiogenesis, our
results may provide another approach to disrupt the
growthoftumors.Thus, by using anavian-specific retro-
virus, wewereabletospecificallytargetthechick vascu-
lature of a growing human medulloblastoma. Even
thoughthe tumorcells remained uninfected by the retro-
virus, we observed suppressed tumor growth demon-
strating the potential therapeutic efficacy of this ap-
In a combination of experiments using retrovirally de-
livered mutant Src and Csk as well as a direct analyses
of src?/?mice, we provide evidence that the Src tyrosine
kinase family distinguishes two pathways of angiogen-
esis. During VEGF-induced angiogenesis, SFK activity
contributes to endothelial cellsurvival. Furthermore, the
VEGF-induced VP is dependent on SFKs, Src, or Yes,
but not Fyn, and the VP response is specific for VEGF
incontrastto inflammation-induced VP.Therefore, while
SFKs serve compensatory roles during embryogenesis
and angiogenesis, VEGF-, but not bFGF-, mediated an-
giogenesis requires Src kinase activity for endothelial
Figure 6. Miles Assay for Vascular Permeability of VEGF in the Skin
of Mice Deficient in Src, Fyn, or Yes
(A)The vascularpermeability properties ofVEGF inthe skin ofsrc?/?
(upper) or src?/?(lower) mice was determined by intradermal injec-
tionofsalineorVEGF (400ng)into mice thathavebeenintravenously
injected withEvan's blue dye.After15min, skinpatches were photo-
graphed (scale bar, 1 mm). Arrowheads indicate the injection sites.
(B) The regions surrounding the injection sites of the VEGF, bFGF,
orsaline were dissected, and the permeability quantitated by elution
oftheEvan's blueinformamideat56?C for24hr, andthe absorbance
measured at 600 nm (left). The ability of an inflammation mediator
(allylisothiocyanate), knownto induce inflammation-related VP, was
tested in src?/?or src?/?mice (right).
(C) The ability of VEGF to induce VP was compared in src?/?, fyn?/?,
or yes?/?mice in the Miles assay. Data for each of the Miles assays
are expressed as the mean ? SD of triplicate animals. src?/?and
yes?/?VP defects compared to control animals were statistically
significant (*p ? 0.05, paired t test), whereas the VP defects in
neither the VEGF-treated fyn?/?mice nor the allyl isothiocyanate±
treated src?/?mice were statistically significant (**p ? 0.05).
these SFKs were shown to be equivalently expressed
in the aortas, skin, and brain of wild-type mice and are
known to be expressed in endothelial cells (Bull et al.,
1994; Kiefer et al., 1994). To our surprise, inflammation-
induced VP was shown to be independent of Src kinase
in these mice, suggesting that the VP activity induced
during inflammationand thatinduced uponVEGF stimu-
lation are regulated by distinct signaling pathways.
Mice lacking pp60c-srcand pp62c-yesshow apparently
VEGF or its receptor die during development. Thus,
VEGF-induced VP activity is not required for develop-
ment. However, it may play a role in wound repair or
goat anti-mouse secondary antibodies as previously described (Eli-
ceiri et al., 1998).
cell survival, whereas VP activity of VEGF depends on
the SFKs, Src, or Yes.
In Vitro Kinase Assay for Src Kinase
The kinase activity of endogenous Src kinase was assayed by the
ability of immunopurified Src to phosphorylate a FAK±GST fusion
protein in an in vitro assay. Src was immunoprecipitated as de-
scribed above and subjected to a kinase assay, and the samples
were analyzed by 15% SDS-PAGE and quantitated as described
previously (Eliceiri et al., 1998).
Antibodies and Reagents
A rabbit polyclonal antibody raised against amino acids 3±18 of
human Src (N-16; Santa Cruz Biotechnology, Santa Cruz, CA) was
used for immunoprecipitation for in vitro kinase assays, and mono-
clonalantibody against avianpp60c-src(Upstate Biotechnology, Lake
Placid, NY) was used for Western blotting as a loading control for
the kinase assays. The Src constructs were obtained from Dr. H.
Varmus (NIH), FAK±GST fusion protein was from Dr. D. Schlaepfer
(The Scripps Research Institute [TSRI]), the DF-1 virus producercell
line was from Dr. D. Foster (Univeristy of Minnesota), the DAOY
medulloblastoma cell line from Dr. W. Laug (Children's Hospital,
USC, Los Angeles), RCAS±GFP was from Dr. C. Cepko (Harvard),
and bFGF was kindly provided by Dr. J . Abraham (Scios, Mountain
View, CA). All other reagents and media were from Sigma-Aldrich
(St Louis, MO) unless otherwise stated.
Immunostaining and Annexin V Labeling of Apoptotic Cells
Cryosections of CAMs treated with RCAS±GFP or RCAS±Src 251
treated with bFGF or VEGF were analyzed for apoptotic cells using
the Apoptag kit (Oncor, Gaithersburg, MD). Sections were also im-
munostained with a rabbit polyclonal anti-vWF (Biogenix, San Ra-
mon, CA)and counterstainedwith1?g/mlDAPI.Fluorescentimages
were captured with a cooled CCD camera (Roper, Trenton, NJ ),
and the fluorescent images were processed and exposure matched
between experimental treatments as previously described (Eliceiri
et al., 1998).
To measure the apoptotic index of retrovirus-infected CAM tis-
sues, FITC-conjugated annexinV (Clontech, Palo Alto, CA)was used
to stain cell suspensions, and the washed cells were analyzed by
flow cytometry. Cell suspensions of CAM cells were prepared from
mock- orvirus-infected CAMs by digestion with 0.1% (w/v) collage-
nase type IV (Worthington Biochemicals, Lakewood, NJ ) in RPMI
1640 of minced CAM tissue rocking for 1 hr at 37?C as previously
described (Brooks et al., 1994b) and filtered through 100 ?M nylon
mesh (Becton Dickinson, Fountain Lakes, NJ ). Fluorescence was
measured with a FACscan flow cytometer (Becton Dickinson) to
count 10,000 cells.
Measurement of vWf staining by FACS was performed with paral-
lel collagenase digested CAM tissue cell preparations, that were
fixed in 1.6% paraformaldehyde, permeabilized in 70% ethanol, in-
cubated the anti-vWf antibody, and detected with a FITC-conju-
gated secondary antibody.
Src Constructs and Retroviruses
For the studies in the chick embryo, the replication competent
RCASBP(A) (Hughes et al., 1987) retrovirus was used to express
mutant Src cDNAs subcloned as NotI±ClaI. These constructs were
transfected into the chicken immortalized fibroblast line, DF-1. Viral
supernatants were collected fromDF-1 producercelllines inserum-
free CLM media. Viral supernatants were concentrated by ultracen-
trifugation at 4?C for 2 hr at 22,000 rpm, and the pellets were resus-
pended in 1/100 the original volume in serum-free media with a titer
of at least 108 i.u. (infectious units)/ml and stored at ?80?C.
For the retrovirus studies in the subcutaneous murine matrigel
angiogenesis assay, GFP, kinase-deleted Src 251, and Csk cDNA
was subcloned into the replication-defective murine Moloney retro-
virus (pLNCX) vector. These constructs were transiently transfected
into the ecotropic producerline to generate cell-free titers of105±106
i.u./ml. Therefore, to increase the effective titer over the 5 day time
course of the angiogenesis assay in the matrigel plug, the virus-
packaging cells expressing the appropriate construct were included
along in the Matrigel to increase the retrovirus infection levels.
Tumor Growth Assay
The 3 and 6 day DAOY medulloblastoma tumor growth assays were
performed in the chick CAM essentially as previously described
(Brooks et al., 1994b). DAOY cells (5 ? 106) were seeded on the
CAM of a 10 day embryo. After7 days, 50 mg tumorfragments were
dissected and reseeded on another 10 day embryo and incubated
for another 3 or 6 days with the topical application (25 ?l) of either
control RCAS±GFP retrovirus, RCAS±Src 251, or mock treatment.
Tumor resections and weighing were performed in a double blind
mannerremoving only the easily definable solid tumormass (Brooks
et al., 1994b). The wet tumor weights after 3 or 6 days were com-
pared with initial weight, and the percent change of tumor weight
was determined for each group.
Recombinant VEGF adenovirus was generated by cloning the hu-
man VEGF cDNA from a human placenta cDNA library into pAd/CI
(J . L., A. Reddy, and D. A. C., unpublished data) and cotransfecting
with pJ M17 into an E1 transcomplementing 293 cell line as pre-
viously described (Bett et al., 1994). High titer virus was isolated,
purified, and titered to 1011pfu/ml as previously described (Chang
et al., 1995). High titer clones were selected based on their expres-
sion of soluble VEGF secreted into the media of COS-7, endothelial
cells, and in chick CAMs infected with the VEGF adenovirus (data
Immunofluorescence and Microscopy
Cryosections of the plugs were also subjected to immunofluores-
cent staining with an anti-CD34 antibody or an anti-flk antibody,
photographed, and quantitated as described above for the CAM
Whole-mount direct fluorescence of RCAS-GFP-infected tumor
fragment was accomplished by dissecting a tumor fragment and
imaging the unfixed tissue directly on a slide with a laser confocal
microscope (MRC 1024; Bio-Rad, Hercules, CA).
Chick Embryo Treatments
Fertilized chick embryos (standard pathogen free grade; SPAFAS,
Preston, CT) were prepared, and the CAM was exposed as pre-
viously described(Eliceirietal., 1998).Forgrowthfactor±only experi-
ments, cortisone acetate±soaked filter disks were soaked with 250
ng of bFGF or VEGF for 2 hr before harvest. For virus experiments
on the CAM, disks were soaked in 20 ?l of viral stock per disk.
These disks were applied to the CAM of 9 day chick embryos and
incubated at 37?C for24 hr. Then, eitherserum-free media orgrowth
factors were added at a concentration of 5 ?g/ml to the CAM in 20
?l of the virus stock as an additional boost of virus to the CAM
tissueandincubated foranadditional72hr.CAM assays was quanti-
tated by counting branch points as described previously (Eliceiri et
al., 1998) in triplicate samples in a double blind manner.
Murine Matrigel Angiogenesis Assay
mented with PBS, bFGF (400 ng/ml), or VEGF (400 ng/ml) (Passaniti
et al., 1992) and murine-specific ecotropic packaging cells (φNX-
Eco; G. Nolan, Stanford) producing retrovirus expressing GFP, Src
251, orCsk cDNAs was injected subcutaneously in6-week-old male
athymic wehi (nu/nu) mice. The plugs remained palpable for5 days,
facilitating a directresectionofthe plug forfurtheranalysis by immu-
noblotting ofplug homogenates orimmunostaining ofplug cryosec-
tions. The accuracy of the quantitative methods was confirmed by
spectrophotometric analysis of homogenates of plugs from animals
Immunoprecipitation and Immunoblotting
CAM tissues were homogenized in a RIPA lysis buffer, used for
immunoprecipitations or immunoblots as previously described (Eli-
ceiri et al., 1998). Anti-Src and anti-flk1 antibodies used in immu-
moblots were detected with horseradish peroxidase-conjugated
Src Requirement for Angiogenesis and Permeability
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Intradermal Ear Injections and Miles Assay
pp60c-src-, pp59c-fyn-, and pp62c-yes-deficient mice (129/Sv/Ev ?
C57Bl6/J ) were generated as previously described (Soriano et al.,
1991) and were the generous gift of Drs. P. Soriano and P. Stein.
Additional stocks were obtained from J ackson Labs. Mouse ears
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injecting 10 ?l of VEGF (400 ng/ml), allyl isothiocyanate (mustard
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previously been intravenously injected with 100 ?l of 0.5% Evan's
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Intracerebral Injection and Determination
of Blood±Brain Barrier Disruption
Saline orVEGF (200 ng in 2 ?l) was injected stereotactically into the
left or right frontal lobe 92 mm to the left/right of the midline, 0.5
mm rostral from bregma, and 3 mm in depth from the dura, respec-
tively. The animals received an Evan's blue solution intravenously
30 min after injection, as described above. After an additional 30
min, the mice were perfused and the brains were removed. Evan's
blue fluorescence was observed using confocal laser microscopy
of fresh unfixed cryosections of the brain.
We thank Tessa Brodhag and Catherine Andrews for expert techni-
cal assistance and Drs. Harold Varmus, Peter K. Vogt, and Bing
J iang for helpful discussions, Dr. K. Spencer (TSRI) for the anti-
vWF immunofluorescence, R. Xiang and C. Dolman for intravenous
injections (TSRI), Archana Reddy for the VEGF adenovirus (TSRI),
and Ana Venegas (NIH) for assistance with mouse breeding. We
also thank Drs. R. Reisfeld (TSRI) and H. Varmus (NIH) for critical
reading of this manuscript. Chick CAM and mouse experiments
were conducted inaccordance withinstitutionaland NIH guidelines.
B. P. E. was supported by an NIH NRSA postdoctoral fellowship
(1F32HL09435), R. P. by Deutsche Forschungs Gemeinschaft Pa
749/1±1, J . D. H. by an NIH training grant (1T32CA75924), J . L. by
an Army Breast Cancer Program (DAMD179616104), and D. A. C.
by grants CA50286, CA45726, HL54444, and P01 CA78045 from the
NIH. This is manuscript 11851-IMM from The Scripps Research
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