VEGF is essential for hypoxia-inducible factor-mediated
neovascularization but dispensable for
Sunday Oladipupoa,1, Song Hub,1, Joanna Kovalskia, Junjie Yaob, Andrea Santeforda, Rebecca E. Sohna, Ralph Shohetc,
Konstantin Maslovb, Lihong V. Wangb,d,2, and Jeffrey M. Arbeita,d,2
aUrology Division, Department of Surgery, andcDepartment of Medicine, University of Hawaii, 96813; andbDepartment of Biomedical Engineering and
dSiteman Cancer Center, Washington University in St. Louis, 63110
Edited* by Gregg L. Semenza, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 24, 2011 (received for review January
Although our understanding of the molecular regulation of adult
neovascularization has advanced tremendously, vascular-targeted
therapies for tissue ischemia remain suboptimal. The master
regulatory transcription factors of the hypoxia-inducible factor
(HIF) family are attractive therapeutic targets because they co-
ordinately up-regulate multiple genes controlling neovasculariza-
diated neovascularization. TetON-HIF-1, K14-Cre, and VEGFflox/flox
of concomitant VEGF. HIF-1 inductionfailed to produce neovascula-
rization in TetON-HIF-1:VEGFΔmice despite robust up-regulation of
angiogenin, and PAI-1. In contrast, endothelial sprouting was pre-
served, enhanced, and more persistent, consistent with marked re-
duction in Dll4-Notch-1 signaling. Optical-resolution photoacoustic
microscopy, which provides noninvasive, label-free, high resolu-
tion, and wide-field vascular imaging, revealedthe absence of both
capillary expansion and arteriovenous remodeling in serially im-
aged individual TetON-HIF-1:VEGFΔmice. Impaired TetON-HIF-1:
VEGFΔneovascularization could be partially rescued by 12-O-tetra-
decanoylphorbol-13-acetate skin treatment. These data suggest
that therapeutic angiogenesis for ischemic cardiovascular disease
may require treatment with both HIF-1 and VEGF.
vascular biology|conditional expression|photoacoustic tomography|
ervation and recovery after ischemia. Although tremendous
progress has been made in understanding the molecular circuits
regulating neovascularization in both benign and malignant dis-
ease, our knowledge remains incomplete. Solid tumors evade
angiogenesis inhibitors, and efficacious therapeutic angiogenesis
for tissue ischemia remains a tantalizing prospect.
The hypoxia-inducible factors-1 and -2 (HIF-1 and HIF-2) are
αβ heterodimeric proteins that make crucial contributions to
neovascularization in wound healing, and benign or malignant
diseases (1, 2). HIFs are master regulatory transcription factors
with >100 known, and potentially hundreds more, target genes con-
HIF target genes coordinates neovascularization at several levels
remodeling, and proangiogenic myeloid cell recruitment (7).
Numerous reports suggested that HIF produced a more robust
neovasculature, with more normal structure and function, com-
pared with single angiogenic factor overexpression (2, 8–10).
However, the contribution of individual target genes encoding
vascular functions to the collective HIF-1 neovascular phenotype
is unknown. Here, we tested the hypothesis that VEGF was dis-
pensableforHIF-mediated neovascularization. Weusedaunique
conditional model of adult neovascularization in the skin, the
eovascularization is crucial for solid tumor growth and
metastatic spread, for wound healing, and for tissue pres-
TetON-HIF-1 transgenic mouse (11) that displays multistage
neovascularization, and myeloid cell recruitment and retention in
the absence of disease. TetON-HIF-1 mice were engineered for
germ-line Cre-mediated VEGF deletion in the same basal kera-
tinocytes targeted for conditional adult HIF-1α induction. VEGF
deletion abrogated neovascularization despite coordinate up-
regulation of multiple angiogenic growth factors. Surprisingly,
endothelial sprouting was activated after HIF-1 induction; how-
ever, these sprouts were nonproductive for neovessel develop-
HIF-1 activation in the absence of VEGF failed to affect vascular
remodeling, andmyeloidcell recruitment wasmarkedly impaired.
Thus, we provide evidence that VEGF is required for HIF-1–
mediated adult neovascularization, but that certain elements of
the angiogenic response proceed without this factor.
TetON-HIF-1:VEGFΔMice Lack Angiogenesis and Vessel Remodeling.
To test the role of VEGF during epithelial HIF-1–induced neo-
vascularization, we combined TetON-HIF-1 mice (11) with K14-
Cre transgenic and VEGF floxed (VEGFf/f) knock-in mice (12–
15). The final genotype of these composite mice was K14-rtTA:
TRE-HIF-1αP402A/P564A/N803A:K14-Cre:VEGFf/f. This genotype,
designated as TetON-HIF-1:VEGFΔ, was n = 12 in the FVB/n
strain. Doxycycline (DOX) treatment of 8- to 12-wk-old TetON-
HIF-1:VEGFf/fmice induced a robust neovascularization that
peaked and plateaued 14 d after initiation (Fig. 1 A and B). In
contrast, neovascularization failed to occur in TetON-HIF-1:
VEGFΔcomposite mice (Fig. 1 A and B). Moreover, contrary to
in TetON-HIF-1:VEGFΔcomposite mice, at baseline before
DOX induction compared with nontransgenic controls, likely due
to differences in tissue fixation/epitope preservation techniques
and endothelial marker antibody detection reagents. Induction of
HIF-1α protein expression was detectable in both interfollicular
both TetON-HIF-1:VEGFΔand TetON-HIF-1:VEGFf/fmice
after 14 days of DOX induction (Fig. 1C).
Temporal control of HIF-1 allowed us to deploy an emerging
technology for noninvasive and label-free functional deter-
mination of microvascular dynamics, optical-resolution photo-
Author contributions: S.O., S.H., K.M., L.V.W., and J.M.A. designed research; S.O., S.H.,
J.K., A.S., and R.E.S. performed research; R.S. and K.M. contributed new reagents/analytic
tools; S.O., S.H., J.K., J.Y., L.V.W., and J.M.A. analyzed data; and S.O., S.H., J.Y., L.V.W., and
J.M.A. wrote the paper.
Conflict of interest statement: L.V.W. has financial interest in Microphotoacoustics, Inc.,
and Endra, Inc., which, however, did not support this work. Other authors declare no
competing financial interest.
*This Direct Submission article had a prearranged editor.
1S.O. and S.H. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 9, 2011
| vol. 108
| no. 32www.pnas.org/cgi/doi/10.1073/pnas.1101321108
acoustic microscopy (OR-PAM) (17, 18). The resolving power
and image segmentation capabilities of OR-PAM enabled mul-
tiparameter determination of vascular morphology including
capillary volume as an indicator of angiogenesis, and arteriove-
nous (arteriolar/venular) volume, vessel length, diameter, and
tortuosity as measures of vascular remodeling. Because OR-
PAM uses hemoglobin for contrast (17), both the imaging and
quantitative segmentation analysis were based on perfused ves-
sels, which, similar to ante mortem lectin perfusion (10), is dis-
tinct from data derived from endothelial marker analysis of
postmortem tissue sections. Serial OR-PAM monitoring of in-
dividual mice from days 0 to 60 of continuous DOX provision
revealed that HIF-1 activation in TetON-HIF-1:VEGFf/fmice
produced an eightfold increase in capillary volume and a 3.5-fold
elevation of arteriovenous and overall vascular volume com-
pared with DOX-induced TetON-HIF-1:VEGFΔor day 0
TetON-HIF-1:VEGFf/fcontrols (Fig. 2 A and Fig. S1B). OR-
PAM segmentation analysis suggested that capillary volume fell
between days 14 and 30 in the TetON-HIF-1:VEGFf/fmice (Fig.
S1). The difference between these data and the microvessel
density analysis based on CD31 decoration of all vessels could be
due to the lack of perfusion of CD31-positive vessels or that OR-
PAM acquires wide field volumetric data and immunofluores-
cence analyzes microvascular area in 2D tissue sections. OR-
PAM also demonstrated abrogation of HIF-1–mediated vasodi-
latation and tortuosity induction in the absence of epithelial
VEGF (Fig. S1B).
Loss of HIF-1–Induced VEGF Produces Sprouting Without Endothelial
the ability to dissect discrete stages of neovascular network de-
velopment and its maintenance due to temporal control of HIF-1
gain of function (11). Here, we tested the necessity of VEGF for
HIF-1 induction of the earliest stages of neovascularization, en-
0 TetON-HIF-1:VEGFf/fmice were used interchangeably in all
Endothelial proliferation remained at barely detectable levels af-
terHIF-1activation in TetON-HIF-1:VEGFΔmice,in contrast to
a robust induction in TetON-HIF-1:VEGFf/fcounterparts (Fig.
S2A). Surprisingly, TetON-HIF-1:VEGFΔmice evidenced a pro-
induction (Fig. 3A, Left and Fig. S2B). In contrast, sprout elabo-
ration in TetON-HIF-1:VEGFf/fwas restricted to the immediate
undetectable by DOX day 14 (Fig. 3A, Left and Fig. S2B). Sprouts
were not detectable in NTG mice at any interval during DOX
treatment (Fig. 3A, Left and Fig. S2B). Because Dll4-Notch1 ac-
signaling via VEGFR2 and VEGFR3 (19–21), we interrogated
mouse ear cross-sections for dual Dll4/CD31 coimmuno-
NTG counterparts up to 30 d of continuous HIF-1 induction (Fig.
3A, Right). In contrast, TetON-HIF-1:VEGFf/fmice evidenced
marked endothelial Dll4 up-regulation after 14 d of HIF-1 in-
duction (Fig. 3, Right Middle). We further explored Notch-1 sig-
naling by determining the expression kinetics and localization of
secretase-mediated receptor cleavage and the active transcrip-
tional Notch component (22). The NICD was detectable in both
the day 14 TetON-HIF-1:VEGFf/fendothelium and in basal ker-
atinocytes (Fig. 3B, arrowheads). In contrast, the NICD was de-
tectable only in a few basal keratinocytes of TetON-HIF-1:
mice compared with 14-d DOX-treated NTG controls (Fig. 3C).
Dll4 and Hey-1 were induced at low, but statistically significant,
levels in TetON-HIF-1:VEGFΔmice compared with NTG con-
trols (Fig. 3C). Collectively, these data suggest that HIF-1 and its
downstream targets (see below) are able to induce endothelial
sprouting, yet these sprouts are nonproductive in forming neo-
the TetON-HIF-1:VEGFΔmice demonstrates that VEGF is re-
VEGF deletion abrogated HIF-1–induced
neovascularization. (A) CD31 immuno-
fluorescence (red) revealed robust neo-
(VEGFf/f) compared with TetON-HIF-1:
VEGFΔ(VEGFΔ) mice, which remained
similar to baseline controls. DAPI was
used as a nuclear counterstain. (B) Quan-
titative analysis of CD31+ vascular area
revealed a threefold elevation in TetON-
HIF-1:VEGFf/fcompared with TetON-HIF-
1:VEGFΔmice induced with doxycycline
for similar time intervals (n = 3–6 mice
per time point). (C) Representative HIF-1α
transgene immunofluorescence indicat-
ing localization to the epidermal and hair
follicle basal cell compartment after 14 d
of DOX induction. (B) VEGFΔor VEGFf/f
data at each DOXday were compared with
VEGFf/fday 0 data by using the unpaired
Student’s t test (**P < 0.01 and ***P <
0.001). (Scale bars: A, 200 μm: C, 50 μm.)
Cre-mediated basal keratinocyte
Oladipupo et al.PNAS
| August 9, 2011
| vol. 108
| no. 32
quired for robust Notch activation in this cellular tissue com-
partment after HIF-1 induction.
Induction of Multiple HIF-1 Angiogenic Target Genes Is Insufficient for
Angiogenesis in the Absence of VEGF. Previous work has high-
lighted the angiogenic potential of several direct and indirect
HIF-1 transcriptional targets (23–29). To determine whether
these targets were induced by HIF-1 in the absence of VEGF,
whole skin extracts were analyzed by using RT-PCR, ELISA, or
antibody microarrays (Fig. 4 A and E). First, we determined the
functional efficiency of keratin-14 regulated Cre-mediated
VEGF deletion. Both mRNA and whole tissue ELISA revealed
baseline or even lower VEGF levels, compared with DOX-
treated NTG controls, despite HIF-1 induction.
Other targets exhibited four expression patterns. PIGF, angio-
genin, MMP-9, PAI-1, and MMP-3 were up-regulated in TetON-
HIF-1:VEGFΔmice at levels comparable with TetON-HIF-1:
VEGFf/fcontrols (Fig. 4F and Fig. S3 A and B). Adrenomedullin
(ADM) and iNOS expression were lower in TetON-HIF-1:
VEGFΔmice on DOX day 3, increased during continuous HIF-1
activation for 14 and 30 d, yet did not reach the levels evident in
TetON-HIF-1:VEGFf/fpositive controls (Fig. 4 B and C). Car-
bonic anhydrase IX (CAIX) progressively increased in TetON-
HIF-1:VEGFΔmice to levels comparable with TetON-HIF-1:
VEGFf/fpositive controls by 30 d of continuous HIF-1 induction
(Fig. 4D). The fourth expression pattern tracked with VEGF ex-
pression, lack or markedly reduced induction in TetON-HIF-1:
VEGFΔmice compared with marked up-regulation in TetON-
counterparts. These molecules, osteopontin
(OPN), pentraxin-3 (PTX-3), cysteine-rich 61/connective tissue
growth factor/nephroblastoma overexpressed (CCN3/NOV), and
activated inflammatory and stromal fibroblasts (30–32). Their
expression pattern was consistent with the marked diminution in
stromal myeloid cell recruitment and retention in TetON-HIF-1:
VEGFΔcompared with TetON-HIF-1:VEGFf/fmice (see below).
Epithelial VEGF Loss Markedly Impairs HIF-1–Mediated Myeloid Cell
Recruitment. HIF-1 is known to induce myeloid cell mobilization,
recruitment, and retention via VEGF, PlGF, and SDF1 (11, 33–36).
As such, we determined myeloid cell recruitment to HIF-1 stim-
ulated skin in the absence of concomitant VEGF. Surprisingly,
there was no significant recruitment of CD45+, CD11b+, or
F4/80+ myeloid, or mast cells in TetON-HIF-1:VEGFΔmice
after 14 d of HIF-1 induction despite significant up-regulation
of the VEGFR1 ligand, PlGF (Figs. 4F and 5 A–C and Fig. S4).
By day 30, a significant increase in both CD45 and CD11b myeloid
cells was achieved in TetON-HIF-1:VEGFΔmice; however, these
myeloid cells were insufficient to facilitate neovascularization in
the absence of epithelial VEGF (Fig. 5 A–C and Fig. S4). Myeloid
cell infiltration after chronic HIF-1 gain of function in DOX day
30 TetON-HIF-1:VEGFΔmice was similar to our previous work
with germ-line skin-targeted constitutive HIF-1 mutants in trans-
genic mice (37). There, stromal myeloid cell trafficking was reg-
ulated by chemokines not VEGF.
12-O-Tetradecanoylphorbol-13-Acetate (TPA)-Induced Inflammation
Produces Extensive Vascular Remodeling with Minimal Microvascular
Density Increase. Topical phorbol ester induces angiogenesis via
enhanced VEGF secretion from the activated epithelium (38,
39). Single-dose TPA treatment produces transient skin changes
resolving after 3–4 d in wild type mice. Previously, we discovered
that germ-line K14-HIF-1P402A/P564Gtransgenic mice responded
to single-dose TPA challenge with a marked and prolonged
stromal and intraepithelial neutrophil infiltrate persisting for 3
wk (37). Neutrophil recruitment was secondary to HIF-1–en-
hanced NFκB signaling and NFκB chemokine target gene up-
regulation by transgenic keratinocytes. We capitalized on this
strategy to determine whether the lack of HIF-1–mediated neo-
vascularization in TetON-HIF-1:VEGFΔ
be rescued. TetON-HIF-1:VEGFΔand TetON-HIF-1:VEGFf/f
were DOX-induced for 14 d to prime the keratinocytes and
stroma and emulate germ-line constitutive activation. One dose
of topical TPA was followed by ear harvest 10 d later. As re-
ported, TPA treatment produced a marked recruitment and re-
tention of CD45 stromal myeloid cells in TetON-HIF-1:VEGFf/f
mice (37), without affecting the frequency of CD11b macrophages
(Fig. S5 A and B). Both TPA-treated DOX day 14 TetON-HIF-1:
VEGFf/fand TetON-HIF-1:VEGFΔmice ear epidermis was
similarly punctuated by microabscesses filled with neutrophils
(Fig. S6, Right), an observation consistent with our previous
findings (37). Moreover, TPA did not further augment VEGF
expression or neovascularization in TetON-HIF-1:VEGFf/fmice
(Fig. 6 A and B). In contrast, single-dose TPA in TetON-HIF-1:
VEGFΔmice produced a twofold increase in VEGF expression
associated with a similar fold increase in neovessel area. TPA
also produced a similar influx of neutrophils in TetON-HIF-1:
VEGFΔmice (Fig. S5A, Left and B, Upper). Partial rescue of
performed in the same VEGFf/for VEGFΔtransgenic mouse serially for 60 d (days 0–30 are represented). Increased capillary density (red asterisk) is evident
particularly by day 14, whereas arteriovenous remodeling (increase in vessel diameter and tortuosity) is detectable at day 14 and prominent by day 30 (blue
arrow) (see also Fig. S1). (Scale bar: 500 μm.)
TetON-HIF-1:VEGFΔmice lacked vessel remodeling. (A) Angiogenesis determination and accompanying vascular remodeling by OR-PAM. OR-PAM was
| www.pnas.org/cgi/doi/10.1073/pnas.1101321108Oladipupo et al.
HIF-1–mediated neovascularization in TetON-HIF-1:VEGFΔ
mice was associated with expansion of an epidermal population
of unrecombined VEGFf/falleles and abscess-derived neutro-
phils (Fig. S5B). However, VEGF expression was undetectable in
the majority of stromal neutrophils not associated with intra-
epidermal abcesses. TPA treatment also produced a statistically
significant increase in capillary and total vessel volume, and
a trend towards an increase in vessel tortuosity, as determined by
OR-PAM (Fig. S7 A and B).
In this study, we used a conditional mouse model to discover that
VEGF, among a repertoire of induced angiogenic target genes,
was necessary for HIF-1–mediated neovascularization. Targeting
able to use unfurred ear skin, enabling visual assessment of neo-
vascular progression. The ear possessed a stereotypic histological
architecture with a sharply delineated boundary between the
transgene expressing basal keratinocytes and the underlying neo-
vessel and inflammatory cell containing stroma. These features
allowed us to determine alterations in tissue molecular signaling
and immunofluorescent localization of induced signaling events
covering the entire spectrum of neovascularization, from initial
endothelial cell activation to development and maintenance of
a complete neovascular network. This combination of tissue or-
ganization for molecular expression localization and immediate
tissue sampling of an activated vasculature was distinct from dis-
ease models that required a lag phase to allow tumors to grow or
ischemic neovascularization to become established. Other studies
used adenoviral expression systems to create controlled time-
dependent ear neovascularization (40). This approach had two
challenges. First, there was an abrupt and massive overexpression
of angiogenic factors followed by rapid decline. Second, viral
particles were deposited in the stroma, an approach that did not
model angiogenic signaling from an activated epithelium that,
component of multistage epithelial carcinogenesis (41).
As expected, we detected DOX-induced up-regulation of mul-
tiple angiogenic target genes that could have compensated for
VEGF deficiency in TetON-HIF-1:VEGFΔmice. A lead example
was the indirect HIF target PlGF. PlGF binds and activates
VEGFR1 and induces phosphorylation of a repertoire of target
VEGFΔmice. (A) Representative confocal CD31 immunofluorescence showing
vessel sprouting in TetON-HIF-1:VEGFΔand TetON-HIF-1VEGFf/fmice (arrows;
Left). Note multiple sprouts in VEGFΔmice at DOX day 14 compared with
their absence in VEGFf/fmice. Representative dual Dll4/CD31 immunofluo-
rescence (Right). VEGFf/fmice showed marked endothelial Dll4 upregulation
from 3 d of HIF-1 induction compared to VEGF δ mice and NTG counterparts.
Sprouts were not detectable in DOX-treated NTG mice (Left). (B) Represen-
tative immunofluorescence of cleaved Notch1 intracellular domain (NICD,
Val1744) in TetON-HIF-1:VEGFΔand TetON-HIF-1VEGFf/fmice (arrows; Middle
and Bottom). (C) RT-PCR analysis (arbitrary units normalized to histone 3.3A)
of the Notch1 ligand Dll4 and downstream target Hey1 showing marked 7-
to 10-fold elevation in day 14 VEGFf/fmice (n = 3–4 mice per time point). In
contrast, Dll4 and Hey-1 were induced at low, albeit statistically significant
levels in VEGFΔmice. Data are mean ± SD. Statistical analysis was the un-
paired Student t test comparing each VEGFΔor VEGFf/fDOX treatment day
to comparably treated NTG controls: *P < 0.05, **P < 0.01 and ***P < 0.001.
(Scale bars: A, Left, 20 μm; A, Right, 200 μm; B, 50 μm.)
Vessel sprouting increase with low-level increase of Notch activity in
mice. (A–D) RT-PCR determination (arbitrary unit normalized against histone
3.3A) of differential patterns of HIF-1 target gene mRNA expression. Adre-
nomedullin (ADM) and iNOS increased 2.4-fold by day 3 in VEGFΔmice, but
further increased by sevenfold during continuous HIF-1 induction for 14 and
30 d, yet did not reach the induction levels of VEGFf/fpositive controls. Sim-
ilarly, carbonic anhydrase IX (CAIX) progressively increased in VEGFΔmice to
levels comparable to TetON-HIF-1:VEGFf/fpositive controls by day 30 (n = 3–4
mice per time point). (E) Whole tissue ELISA revealed baseline or lower VEGF
levels in VEGFΔmice, compared with DOX-treated NTG controls, during HIF-1
induction (n = 3 mice per time point). (F) PIGF was up-regulated in VEGFΔ
mice at similar levels as VEGFf/fcontrols (n = 3 mice per time point). Data
analyzed as in Fig. 1: *P < 0.05, **P < 0.01, and ***P < 0.001.
Induction of multiple HIF-1 angiogenic targets in TetON-HIF-1:VEGFΔ
Oladipupo et al.PNAS
| August 9, 2011
| vol. 108
| no. 32
has been shown to amplify or inhibit VEGF signaling through
for overexpression in basal keratinocytes (23), or after local ade-
deletion or antibodies targeting PlGF inhibited tumor growth via
multiple mechanisms, including inhibition of angiogenesis, mye-
models of tissue ischemia, PlGF was required for vascular di-
latation and collateralization (24, 46). Thus, we were surprised
that despite robust PlGF induction, angiogenesis, vascular di-
latation, arteriovenous remodeling, and myeloid cell recruitment
were unaffected by HIF-1 induction in the absence of VEGF. As
function of PlGF, but is in contrast to “direct” angiogenesis in-
duction of prior skin and adenoviral studies. The VEGF domi-
nance in our model is also in line with recent studies questioning
the contribution of PlGF to angiogenesis (47, 48).
In addition to PlGF, other angiogenic targets and vascu-
lar remodeling molecules were up-regulated in TetON-HIF-1:
VEGFΔmice. The HIF target adrenomedullin was shown to pro-
mote physiological and pathological angiogenesis in vitro and in
animal models (49–51). ADM signals angiogenesis by binding
calcitonin-receptor-like receptor, which is widely expressed on
normal and hypoxic endothelial cells (49, 52). Angiogenin was
another molecule previously demonstrated to be angiogenic, yet
without consequence in TetON-HIF-1:VEGFΔmice (28, 29). In
addition to these direct proangiogenic HIF targets, molecules
modulation, MMP9, MMP3, and PAI-1, and vascular dilatation,
iNOS, were also up-regulated in TetON-HIF-1:VEGFΔmice.
Thus, many of the molecules in the angiogenic “toolbox” were el-
evated by HIF-1, yet all required coordinate VEGF up-regulation
to affect neovascularization and its modulation. Our data provide
additional insight into studies wherein deletion of the VEGF HIF
VEGF gene deletion in keratinocytes diminished tumor growth
in each of these studies, variable levels of neovascularization were
present, likely from compensatory angiogenic factors. Our use of
topical TPA challenge with its attendant prolonged stromal in-
flammation and neovascularization rescue emulated disease-as-
sociated angiogenic compensation. We were surprised that the
VEGF increment induced by TPA in TetON-HIF-1:VEGFΔ
mice was not due to the numerous neutrophils infiltrating
throughout the stroma. Rather, the VEGF increment was local-
ized to the epidermis, likely due to expansion of a small pop-
ulation of preexisting basal keratinocytes with unrecombined
VEGFf/falleles and to focal neutrophil accumulations beneath
intraepidermal abscesses. These data didnot rule out aneovessel
activation function for the stromal neutrophils, which have been
shown in previous studies to be sources of compensatory angio-
genic factors such as FGF-2 in tumors during VEGF signaling
blockade (54). As such, our conditional model enabled precise
genetic dissection of “intrinsic” HIF-1 functions while enabling
titration of pathophysiological processes to test for complemen-
tation of baseline neovascularization deficiencies.
Finally, ear skin allowed us to continue to use an emerging
technique for determination of neovascular architecture and
microvessel function photoacoustic microscopy (PAM) (17).
PAM and the derivative used in our study, OR-PAM, use red
blood cell (RBC) hemoglobin for endogenous contrast. Dif-
fraction-limited optical focusing and exquisite (100%) sensitivity
to optical absorption enables single RBC resolution and capillary
resolution in vivo (18). Additionally, the wide field of view of
OR-PAM enabled us to determine the necessity of VEGF for
both angiogenesis and arteriovenous remodeling in the context
of HIF-1 induction. These features plus simultaneous deter-
mination of multiple parameters of neovascular function and
tissue metabolism will be a boon for future studies in models of
neovascular disease (17).
In conclusion, we demonstrate that despite induction of multi-
ple angiogenic target genes, VEGF is necessary for HIF-1 medi-
ated neovascularization. The VEGF requirement can be sub-
stantially, although not entirely, rescued by chemokine-activated
Recent studies have demonstrated provocative linkage between
age, cardiovascular disease, and insufficiency of HIF-1 expression
in both ischemic tissues and infiltrating myeloid cells (55, 56).
Because aging can also impair induction of VEGF expression
(57, 58), our work suggests that therapeutic angiogenesis may re-
quire combinatorial restoration or administration of both HIF-1
and VEGF to achieve optimal tissue neovascularization.
Materials and Methods
Mouse Development, Target Expression Analysis, Photoacoustic Microscopy,
and Inflammation Challenge. Detailed description of the mouse intercrosses to
generate target and control genotypes, tissue molecular and expression
analysis, photoacoustic microscopy, and inflammation challenge experi-
ments are available in SI Materials and Methods. The Animal Studies Com-
mittee of Washington University in St. Louis approved all animal care and
of VEGF. (A) Representative CD31/CD11b coimmunofluorescence showing
marked temporal delay and diminution of stromal myeloid cell infiltration
in VEGFΔmice compared with early and robust CD11b+ myeloid cell re-
cruitment inVEGFf/fcounterparts. (B and C) Quantification of stromal area
occupied by CD11b+ (B) (n = 3–4 mice per time point) and CD45+ cells (C; see
Fig. S5 for representative immunofluorescent images) (n = 3–6 mice per time
point). Data analyzed as in Fig. 1: *P < 0.05, **P < 0.01, and ***P < 0.001.
(Scale bar: 200 μm.)
HIF-1–mediated myeloid cell recruitment is abrogated in the absence
rescues neovascularization and results in moderate VEGF induction. (A)
Quantification of CD31(+) area in (n = 3–6 mice per time point) revealed
a twofold increase in neovascularization in TPA-treated VEGFΔmice. (B)
Whole ear tissue ELISA for VEGF. A single dose of TPA in VEGFΔmice pro-
duced a twofold increase in VEGF. In contrast, VEGF levels were not further
increased in TPA-treated VEGFf/fmice. (n = 3–6 mice per time point). Data
analyzed as in Fig. 1: *P < 0.05, **P < 0.01, and ***P < 0.001.
12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment partially
| www.pnas.org/cgi/doi/10.1073/pnas.1101321108Oladipupo et al.
Statistical Analysis. The data are reported as the mean ± SD. Data from DOX- Download full-text
treated TetON-HIF-1:VEGFΔor TetON-HIF-1VEGFf/fmice were compared ei-
ther with day 0 or NTG mice treated with DOX for the same interval by using
the unpaired Student t test (GraphPad Prism 5). There were 3–6 mice per
group unless otherwise indicated.
ACKNOWLEDGMENTS. We thank Hans Peter Gerber and Napoleon Ferrara
for the gift of the VEGFflox/floxmice, Rick Bruick for the gift of the TRE-HIF-
1αP402A/P464A/N803Aplasmid for construction of the TRE-HIF-1αP402/464A/N803A
transgenic mice, and Adam Glick for the gift of the K5-rtTA transgenic mice.
This work was supported by National Institutes of Health Grants R01-
CA90722, R01 EB000712, R01 NS46214, R01 EB008085, and U54 CA136398,
and the Beatrice Roe Urologic Cancer Fund.
1. Hickey MM, Simon MC (2006) Regulation of angiogenesis by hypoxia and hypoxia-
inducible factors. Curr Top Dev Biol 76:217–257.
2. Rey S, Semenza GL (2010) Hypoxia-inducible factor-1-dependent mechanisms of vas-
cularization and vascular remodelling. Cardiovasc Res 86:236–242.
3. Wenger RH, Stiehl DP, Camenisch G (2005) Integration of oxygen signaling at the
consensus HRE. Sci STKE 2005:re12.
4. Ortiz-Barahona A, Villar D, Pescador N, Amigo J, del Peso L (2010) Genome-wide
identification of hypoxia-inducible factor binding sites and target genes by a proba-
bilistic model integrating transcription-profiling data and in silico binding site pre-
diction. Nucleic Acids Res 38:2332–2345.
5. Mole DR, et al. (2009) Genome-wide association of hypoxia-inducible factor (HIF)-
1alpha and HIF-2alpha DNA binding with expression profiling of hypoxia-inducible
transcripts. J Biol Chem 284:16767–16775.
6. Benita Y, et al. (2009) An integrative genomics approach identifies Hypoxia Inducible
Factor-1 (HIF-1)-target genes that form the core response to hypoxia. Nucleic Acids
7. Semenza GL (2010) Vascular responses to hypoxia and ischemia. Arterioscler Thromb
Vasc Biol 30:648.
8. Kelly BD, et al. (2003) Cell type-specific regulation of angiogenic growth factor gene
expression and induction of angiogenesis in nonischemic tissue by a constitutively
active form of hypoxia-inducible factor 1. Circ Res 93:1074–1081.
9. Hirota K, Semenza GL (2006) Regulation of angiogenesis by hypoxia-inducible factor
1. Crit Rev Oncol Hematol 59:15–26.
10. Elson DA, et al. (2001) Induction of hypervascularity without leakage or inflammation
in transgenic mice overexpressing hypoxia-inducible factor-1alpha. Genes Dev 15:
11. Oladipupo SS, et al. (2011) Conditional HIF-1 induction produces multistage neo-
vascularization with stage-specific sensitivity to VEGFR inhibitors and myeloid cell
independence. Blood 117:4142–4153.
12. Bekeredjian R, et al. (2010) Conditional HIF-1alpha expression produces a reversible
cardiomyopathy. PLoS ONE 5:e11693.
13. Diamond I, Owolabi T, Marco M, Lam C, Glick A (2000) Conditional gene expression in
the epidermis of transgenic mice using the tetracycline-regulated transactivators tTA
and rTA linked to the keratin 5 promoter. J Invest Dermatol 115:788–794.
14. Jonkers J, et al. (2001) Synergistic tumor suppressor activity of BRCA2 and p53 in
a conditional mouse model for breast cancer. Nat Genet 29:418–425.
15. Gerber HP, et al. (1999) VEGF is required for growth and survival in neonatal mice.
16. Rossiter H, et al. (2004) Loss of vascular endothelial growth factor a activity in murine
epidermal keratinocytes delays wound healing and inhibits tumor formation. Cancer
17. Wang LV (2009) Multiscale photoacoustic microscopy and computed tomography.
Nat Photonics 3:503–509.
18. Maslov K, Zhang HF, Hu S, Wang LV (2008) Optical-resolution photoacoustic micros-
copy for in vivo imaging of single capillaries. Opt Lett 33:929–931.
19. Tammela T, et al. (2008) Blocking VEGFR-3 suppresses angiogenic sprouting and
vascular network formation. Nature 454:656–660.
20. Thurston G, Noguera-Troise I, Yancopoulos GD (2007) The Delta paradox: DLL4 blockade
leads to more tumour vessels but less tumour growth. Nat Rev Cancer 7:327–331.
21. Hellström M, et al. (2007) Dll4 signalling through Notch1 regulates formation of tip
cells during angiogenesis. Nature 445:776–780.
22. Kopan R, Ilagan MXG (2009) The canonical Notch signaling pathway: Unfolding the
activation mechanism. Cell 137:216–233.
23. Odorisio T, et al. (2002) Mice overexpressing placenta growth factor exhibit increased
vascularization and vessel permeability. J Cell Sci 115:2559–2567.
24. Luttun A, et al. (2002) Revascularization of ischemic tissues by PlGF treatment, and inhibi-
tion of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med 8:831–840.
25. Roy H, et al. (2005) Adenovirus-mediated gene transfer of placental growth factor to
perivascular tissue induces angiogenesis via upregulation of the expression of en-
dogenous vascular endothelial growth factor-A. Hum Gene Ther 16:1422–1428.
26. Abe M, et al. (2003) Adrenomedullin augments collateral development in response to
acute ischemia. Biochem Biophys Res Commun 306:10–15.
27. Oehler MK, Hague S, Rees MC, Bicknell R (2002) Adrenomedullin promotes formation
of xenografted endometrial tumors by stimulation of autocrine growth and angio-
genesis. Oncogene 21:2815–2821.
28. Raghu H, et al. (2010) Suppression of uPA and uPAR attenuates angiogenin mediated
angiogenesis in endothelial and glioblastoma cell lines. PLoS ONE 5:e12458.
29. Yoshioka N, Wang L, Kishimoto K, Tsuji T, Hu GF (2006) A therapeutic target for
prostate cancer based on angiogenin-stimulated angiogenesis and cancer cell pro-
liferation. Proc Natl Acad Sci USA 103:14519–14524.
30. Mantovani A, Garlanda C, Doni A, Bottazzi B (2008) Pentraxins in innate immunity:
From C-reactive protein to the long pentraxin PTX3. J Clin Immunol 28:1–13.
31. Keeley EC, Mehrad B, Strieter RM (2008) Chemokines as mediators of neo-
vascularization. Arterioscler Thromb Vasc Biol 28:1928–1936.
32. Scatena M, Liaw L, Giachelli CM (2007) Osteopontin: A multifunctional molecule
regulating chronic inflammation and vascular disease. Arterioscler Thromb Vasc Biol
33. Ceradini DJ, et al. (2004) Progenitor cell trafficking is regulated by hypoxic gradients
through HIF-1 induction of SDF-1. Nat Med 10:858–864.
34. Grunewald M, et al. (2006) VEGF-induced adult neovascularization: Recruitment, re-
tention, and role of accessory cells. Cell 124:175–189.
35. Hattori K, et al. (2002) Placental growth factor reconstitutes hematopoiesis by re-
cruiting VEGFR1(+) stem cells from bone-marrow microenvironment. Nat Med 8:
36. Fischer C, et al. (2007) Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors
without affecting healthy vessels. Cell 131:463–475.
37. Scortegagna M, et al. (2008) HIF-1alpha regulates epithelial inflammation by cell
autonomous NFkappaB activation and paracrine stromal remodeling. Blood 111:
38. Kishimoto J, et al. (2000) In vivo detection of human vascular endothelial growth
factor promoter activity in transgenic mouse skin. Am J Pathol 157:103–110.
39. Morris PB, Hida T, Blackshear PJ, Klintworth GK, Swain JL (1988) Tumor-promoting
phorbol esters induce angiogenesis in vivo. Am J Physiol 254:C318–C322.
40. Nagy JA, Shih SC, Wong WH, Dvorak AM, Dvorak HF (2008) Chapter 3. The adenoviral
vector angiogenesis/lymphangiogenesis assay. Methods Enzymol 444:43–64.
41. Hanahan D, Folkman J (1996) Patterns and emerging mechanisms of the angiogenic
switch during tumorigenesis. Cell 86:353–364.
42. Park JE, Chen HH, Winer J, Houck KA, Ferrara N (1994) Placenta growth factor. Po-
tentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and
high affinity binding to Flt-1 but not to Flk-1/KDR. J Biol Chem 269:25646–25654.
43. Landgren E, Schiller P, Cao Y, Claesson-Welsh L (1998) Placenta growth factor stim-
ulates MAP kinase and mitogenicity but not phospholipase C-gamma and migration
of endothelial cells expressing Flt 1. Oncogene 16:359–367.
44. Eriksson A, et al. (2002) Placenta growth factor-1 antagonizes VEGF-induced angio-
genesis and tumor growth by the formation of functionally inactive PlGF-1/VEGF
heterodimers. Cancer Cell 1:99–108.
45. Autiero M, et al. (2003) Role of PlGF in the intra- and intermolecular cross talk be-
tween the VEGF receptors Flt1 and Flk1. Nat Med 9:936–943.
46. Pipp F, et al. (2003) VEGFR-1-selective VEGF homologue PlGF is arteriogenic: Evidence
for a monocyte-mediated mechanism. Circ Res 92:378–385.
47. Van de Veire S, et al. (2010) Further pharmacological and genetic evidence for the
efficacy of PlGF inhibition in cancer and eye disease. Cell 141:178–190.
48. Bais C, et al. (2010) PlGF blockade does not inhibit angiogenesis during primary tumor
growth. Cell 141:166–177.
49. Nikitenko LL, et al. (2006) Adrenomedullin and CGRP interact with endogenous cal-
citonin-receptor-like receptor in endothelial cells and induce its desensitisation by
different mechanisms. J Cell Sci 119:910–922.
50. Ishikawa T, et al. (2003) Adrenomedullin antagonist suppresses in vivo growth of
human pancreatic cancer cells in SCID mice by suppressing angiogenesis. Oncogene
51. Shindo T, et al. (2001) Vascular abnormalities and elevated blood pressure in mice
lacking adrenomedullin gene. Circulation 104:1964–1971.
52. Nikitenko LL, Smith DM, Bicknell R, Rees MC (2003) Transcriptional regulation of the
CRLR gene in human microvascular endothelial cells by hypoxia. FASEB J 17:
53. Tsuzuki Y, et al. (2000) Vascular endothelial growth factor (VEGF) modulation by
targeting hypoxia-inducible factor-1alpha—> hypoxia response element—> VEGF
cascade differentially regulates vascular response and growth rate in tumors. Cancer
54. Casanovas O, Hicklin DJ, Bergers G, Hanahan D (2005) Drug resistance by evasion of
antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors.
Cancer Cell 8:299–309.
55. Bosch-Marce M, et al. (2007) Effects of aging and hypoxia-inducible factor-1 activity
on angiogenic cell mobilization and recovery of perfusion after limb ischemia. Circ
56. Rey S, et al. (2009) Synergistic effect of HIF-1alpha gene therapy and HIF-1-activated
bone marrow-derived angiogenic cells in a mouse model of limb ischemia. Proc Natl
Acad Sci USA 106:20399–20404.
57. Sadoun E, Reed MJ (2003) Impaired angiogenesis in aging is associated with alter-
ations in vessel density, matrix composition, inflammatory response, and growth
factor expression. J Histochem Cytochem 51:1119–1130.
58. Iemitsu M, Maeda S, Jesmin S, Otsuki T, Miyauchi T (2006) Exercise training improves
aging-induced downregulation of VEGF angiogenic signaling cascade in hearts. Am J
Physiol Heart Circ Physiol 291:H1290–H1298.
Oladipupo et al.PNAS
| August 9, 2011
| vol. 108
| no. 32