Leptin induces vascular permeability and
synergistically stimulates angiogenesis
with FGF-2 and VEGF
Renhai Cao*†, Ebba Brakenhielm*, Claes Wahlestedt†, Johan Thyberg‡, and Yihai Cao*§
*Laboratory of Angiogenesis Research, Microbiology and Tumor Biology Center, †Center for Genomic Research, ‡Department of Cell and Molecular Biology,
Karolinska Institute, S-171 77 Stockholm, Sweden
Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved March 15, 2001 (received for review November 29, 2000)
Most endocrine hormones are produced in tissues and organs with
permeable microvessels that may provide an excess of hormones
to be transported by the blood circulation to the distal target
organ. Here, we investigate whether leptin, an endocrine hormone, induces the formation of vascular fenestrations and permeability, and we characterize its angiogenic property in the presence
of other angiogenic factors. We provide evidence that leptininduced new blood vessels are fenestrated. Under physiological
conditions, capillary fenestrations are found in the leptin-producing adipose tissue in lean mice. In contrast, no vascular fenestrations were detected in the adipose tissue of leptin-deficient obyob
mice. Thus, leptin plays a critical role in the maintenance and
regulation of vascular fenestrations in the adipose tissue. Leptin
induces a rapid vascular permeability response when administrated intradermally. Further, leptin synergistically stimulates angiogenesis with fibroblast growth factor (FGF)-2 and vascular
endothelial growth factor (VEGF), the two most potent and commonly expressed angiogenic factors. These findings demonstrate
that leptin has another new function—the increase of vascular
permeability.
tenance and regulation of vascular fenestrations and in permeability in the adipose tissue are not yet fully understood. In most
leptin-producing tissues, including the placenta and the adipose
tissue, other angiogenic factors are also coexpressed (6). Thus,
it is important to investigate the angiogenic features of leptin in
the presence of several other angiogenic factors. Here, we
investigate whether leptin induces the formation of vascular
fenestrations and permeability, and we study its angiogenic
property in the presence of other angiogenic factors.
Materials and Methods
Reagents and Animals. Recombinant human leptin and VEGF165
were obtained from R & D Systems. Recombinant human
FGF-2 was obtained from Scios Nova (Mountain View, CA).
C57BLy6J obyob mice were obtained from M & B AyS, Ry,
Denmark. Male 5- to 6-week-old C57BLy6J and female
BALByc mice were acclimated and caged in groups of six or
fewer. Animals were anesthetized in a methoxyflurane chamber
before all procedures and killed with a lethal dose of CO2. All
animal studies were reviewed and approved by the animal care
and use committee of the Stockholm Animal Board.
leptin u obesity u vascular fenestrations u neovascularization
Endothelial Cell Proliferation Assay. A 72-h bovine capillary endo-
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O
besity is a global public health problem that is associated
with diabetes mellitus, coronary heart disease, tumors, and
sleep breathing problems. A key gene product that regulates
body weight is leptin. Leptin is a circulating hormone that
regulates adipose tissue mass through hypothalamic effects on
satiety and energy expenditure. Recently, leptin was found to
stimulate angiogenesis in animal models (1, 2). Whereas adipose
tissue is considered to be the key site for leptin production, this
hormone is also produced in actively angiogenic tissues such as
the placenta and fetal tissues such as heart, bone, and hair
follicles, which suggests that it may stimulate neovascularization
in these tissues (3). Thus, leptin may act both as an endocrine
hormone and as a paracrine growth factor.
Most endocrine hormones are produced in tissues and organs
with fenestrated endothelium (4) that may provide an excess of
hormones to be transported by blood circulation to the distal
target organ. This function seems to be particularly important for
the adipose tissue because a global control of body weight, food
intake, energy expenditure, and thermogenesis requires release
of both endocrine hormones and small molecules into the
circulatory system. To accomplish these functions, vascular
fenestrations should be regulated in an organ or tissue in such a
way that fenestrations can be switched on and off. Vascular
fenestrations are an important structural basis for vascular
permeability (4, 5)
Several soluble growth factors are found to regulate vascular
fenestrations and permeability. Examples are vascular endothelial growth factoryvascular permeability factor (VEGFyVPF)
and VEGF-C (6, 7). Although these factors participate in the
control of vascular leakage, they have several other angiogenesisrelated functions in vivo (6). However, their roles in the main6390 – 6395 u PNAS u May 22, 2001 u vol. 98 u no. 11
thelial (BCE) cell proliferation assay was performed as described
(8). BCE cells were maintained in DMEM containing 10%
heat-inactivated bovine calf serum (BCS) and 3 ngyml recombinant human FGF-2. Cells growing in gelatinized 6-well plates
were dispersed in 0.05% trypsin solution and resuspended with
DMEM containing 5% BCS. Approximately 10,000 cells in 0.5
ml were added to each gelatinized well of 24-well plates and
incubated at 37°C for 1 h. Leptin and FGF-2 samples in
triplicates were added to cells to a final volume of medium of 1
ml per well. After a 72-h incubation, cells were trypsinized,
resuspended in Isoton II solution (Beckman Coulter AB, Sweden), and counted with a Coulter Counter.
Mouse Corneal Angiogenesis Assay. The mouse corneal assay was
performed according to procedures described (9). Corneal micropockets were created with a modified von Graefe cataract
knife in both eyes of male 5- to 6-week-old C57BLy6J mice. A
micropellet (0.35 3 0.35 mm) of sucrose and aluminum sulfate
coated with Hydron polymer type NCC (Interferon Sciences,
New Brunswick, NJ) containing 160 ng of leptin, 160 ng of
VEGF, or 80 ng of FGF-2 was implanted into each pocket. The
pellet was positioned 0.6–0.8 mm from the corneal limbus. After
implantation, erythromycin ophthalmic ointment was applied to
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor;
BCE, bovine capillary endothelial.
§To
whom reprint requests should be addressed. E-mail: yihai.cao@mtc.ki.se.
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.
www.pnas.orgycgiydoiy10.1073ypnas.101564798
Fig. 1. Endothelial cell growth and corneal angiogenic responses induced by leptin, FGF-2, and VEGF. (e) BCE cells were incubated with various concentrations
of leptin or 1 ngyml FGF-2. After 72 h, cells were counted. Values represent means (6SEM) of triplicate of each sample. Statistically significant values were
obtained at concentrations above 5 nM (P 5 0.085 at 5 nM, P 5 0.0014 at 10 nM, and P 5 0.027 at 25 nM) (a) A cornea of the control group of mice implanted
with sucrose aluminum sulfate and Hydron (without growth factors). Pellets containing sucrose aluminum sulfate, Hydron, and 160 ng of leptin (b), 80 ng of FGF-2
(c), or 160 ng of VEGF (d) were implanted into corneal micropockets of mice. Corneas were photographed with a stereomicroscope on day 5 after pellet
implantation; positions of implanted pellets are indicated by white arrows. Maximal vessel length ( f), clock-hours of circumferential neovascularization (g), and
area of neovascularization (h) are presented as mean determinants (6SEM) of 9 or 10 corneas in each group. P values were calculated according to a standard
two-tailed Student’s t test. ***, P , 0.001.
Immunohistochemistry. The growth factor-implanted mouse eyes
were enucleated at day 6 after implantation and immediately
frozen on dry ice and stored at 280°C before use. Tissue sections
of 14 mm were dissected with a cryostat and immersed in acetone
for 10 min. Tissue slices were washed with PBS, blocked with 3%
BSA in PBS for 20 min, and incubated for 1 h with a monoclonal
rat antibody against mouse CD31 antigen (PharMingen). After
washing with PBS, a secondary FITC-conjugated rabbit anti-rat
IgG was incubated with the tissue sections for 1 h. The immunostained signals were examined under a fluorescence microscope. Corneal microvessels were counted in at least six sections
at 203 magnification.
Electron Microscopy. At day 6 after implantation of angiogenic
factors, the animals were killed and the eyes were removed and
immersed in a fixative containing 3% glutaraldehyde in 0.1 M
sodium cacodylate–HCl buffer (pH 7.3) with 0.05 M sucrose. A
few hours later, the parts of the corneas containing ingrowing
blood vessels were dissected out, cut into small pieces, and put
in fresh fixative. The subcutaneous adipose tissues of C57BLy6J
wild-type mice and obyob mice were removed and immersed in
glutaraldehyde fixative. After rinsing in buffer, the specimens
were postfixed in 1.5% osmium tetroxide in 0.1 M cacodylate
buffer (pH 7.3) with 0.7% potassium ferrocyanate for 2 h at 4°C,
dehydrated in ethanol (70%, 95%, and 100%), stained with 2%
uranyl acetate in ethanol, and embedded in Spurr low-viscosity
epoxy resin. Thin sections were cut with diamond knives on an
LKB Ultrotome IV, picked up on carbon-coated Formvar films,
stained with alkaline lead citrate, and examined in a Philips
CM120 Twin electron microscope at 80 kV.
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Permeability Assay. Female 6-week-old BALByc white mice were
shaved. Four days later, the mice were anesthetized with a
mixture of Hypnorm (Janssen Animal Health, Belgium) and
Cao et al.
Dormicum (Roche) (1:1) in an H2O solution, and 150 ml of 1%
Evans blue dye solution was injected into the tail vein of each
mouse. After 5 min, 50 ng of leptin or VEGF in a volume of 20
ml of PBS was injected at adjacent locations intradermally in the
middorsum of the same animals. The extravasation of Evans blue
dye was recorded with a digital camera for up to 4 h. As controls,
50 ng of BSA or FGF-2 was injected into the same positions in
animals of a separate group. Five animals were used in each of
the treated and control groups.
Statistics. Statistical evaluation of the results was made by a
two-tailed Student’s t test with INSTAT 1.1 and Microsoft EXCEL
5 programs, and by a one-way ANOVA followed by a Newman–
Keuls post hoc test. To relate the effects of individual and
combined treatments, the effects of leptin and FGF-2 applied
alone were summarized and compared with the effects of leptin
and FGF-2 applied together by using the Mann–Whitney U test.
The same method was used to compare the effects of leptin and
VEGF used alone or together.
Results
Capillary Endothelial Cell Growth. To study the effect of leptin on
capillary endothelial cell growth, various concentrations of
leptin were assayed on BCE cell proliferation and migration.
Significant stimulation of BCE cell proliferation was detected at
concentrations above 10 ngyml. Human leptin stimulated BCE
cell proliferation in a dose-dependent manner (Fig. 1e). These
findings indicate that leptin stimulates capillary endothelial cell
proliferation, which is a critical step for angiogenesis.
Angiogenic Responses. We chose the mouse corneal angiogenesis
model to study the structure of microvessels induced by a single
angiogenic agent because the cornea remains avascular under
physiological conditions. The corneal angiogenic response induced by leptin (Fig. 1b) differed from those induced by FGF-2
(Fig. 1c) and VEGF (Fig. 1d). Although the lengths (Fig. 1f ) of
leptin- and VEGF-stimulated blood vessels were similar, the
clock hours (Fig. 1g) and areas (Fig. 1h) of VEGF-induced
vessels were significantly greater compared with the leptinPNAS u May 22, 2001 u vol. 98 u no. 11 u 6391
MEDICAL SCIENCES
each eye. The eyes were examined by a slit-lamp biomicroscope
on day 5 or day 6 after pellet implantation. Vessel length
and clock-hours of circumferential neovascularization were
measured.
Fig. 3. Details of capillary structures in adipose tissue from (a) wild-type (wt)
and (b) obyob mice. At least 200 capillary sections were examined in each
group. Black arrows point to endothelial fenestrations. Arrowheads mark
caveolae. A, adipocyte; L, capillary lumen; RB, red blood cell; E, endothelial
cell; M, collagenous matrix of the cornea. (Bars 5 0.2 mm.)
Fig. 2. Thin parts of the walls of microvessels growing into the mouse
cornea after stimulation with VEGF (a), leptin (b and c), FGF-2 (d) and FGF-2
1 leptin (e). Arrowheads mark endothelial fenestrations. L, capillary lumen; M, collagenous matrix of the cornea; P, perivascular cell; E, endothelial cell. (Bars 5 0.2 mm.)
stimulated angiogenesis. In FGF-2-implanted corneas, new microvessels crossed the cornea from the limbus toward the pellet,
occasionally penetrating into the pellet (Fig. 1c). In addition,
microvessels induced by all three factors were dilated (Fig. 1
b–d). The angiogenic response stimulated by VEGF was intense,
with a high density of capillary sprouts that seemed to be leaky
and tended to form capillary blobs at the growing edge (Fig. 1d
and Fig. 5c). We should emphasize that 160 ng of leptin, 80 ng
of FGF-2, or 160 ng of VEGF was used for implantation into
each cornea. According to other published results and our
growth factor titration experiments, the amounts of FGF-2 and
VEGF used here are minimal doses that produce maximal
effects of corneal angiogenesis in C57BLy6J mice (10). We have
found that, similar to VEGF, 160 ng of leptin induced the
maximal growth of corneal angiogenesis and that higher doses
did not significantly increase the angiogenic potency.
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Capillary Fenestrations of Newly Formed Blood Vessels. The differ-
ences in the corneal angiogenesis responses induced by leptin,
FGF-2, and VEGF prompted us to study the ultrastructure of
these microvessels. Corneal vessels were examined by electron
microscopy and were found to be completely capillary in nature.
They consisted of single layers of endothelial cells occasionally
surrounded by pericytes (Fig. 2 c and e). No smooth muscle cells
were evident, and in most locations, the endothelium was in
direct contact with the dense collagenous matrix of the cornea.
The formation of endothelial fenestrations was found in leptininduced capillaries (Fig. 2 b and c arrowheads). Although their
frequency of occurrence varied, fenestrations could often be
6392 u www.pnas.orgycgiydoiy10.1073ypnas.101564798
found in the thinnest parts of the capillary walls. Notably,
VEGF-induced capillaries demonstrated numerous fenestrations that had diameters of about 50 nm and usually contained
fine diaphragms (Fig. 2a arrowheads). Their frequency of occurrence varied, but up to 2 or 3 fenestrations could be found per
mm of blood vessel cross section. In striking contrast to the above
findings, no fenestrations were detected in capillaries induced by
FGF-2 (Fig. 2d). Interestingly, leptin facilitated the formation of
a few endothelial fenestrations in microvessels induced by FGF-2
when both factors were used to stimulate angiogenesis (Fig. 2e).
These data demonstrate that leptin and VEGF, but not FGF-2,
induce vascular fenestrations in newly formed blood vessels.
Capillary Fenestrations in Adipose Tissues. Capillary fenestrations
were examined using subcutaneous adipose tissue derived from
wild-type and obyob C57BLy6J mice. In general, the microvessel density in the adipose tissue seemed to be lower in obyob
mice than in wild-type mice. The reduction of microvessel
density could be caused by the increased size of adipocytes in
obyob mice. In wild-type mice, a small number of fenestrated
capillaries were detected. In some cases, several fenestrations
were present in a single endothelial cell section (Fig. 3). When
the thickness of the sections was taken into account (50 nm per
section), one or more fenestrations were estimated to occur
every 1.5–2 mm along the length of the capillaries. This frequency
seemed to be lower than that observed in the corneal vessels.
However, this fact was not surprising, because the corneal blood
vessels were exposed to higher doses of leptin stimulation than
were the capillaries of the normal adipose tissue. In contrast to
the wild-type mice, no fenestrations were found in the adipose
tissue of obyob mice. These data indicate that leptin plays a
significant role in the maintenance of vascular fenestrations in
the adipose tissue under physiological conditions.
Vascular Permeability. Vascular fenestration is one of the struc-
tural bases that cause capillary permeability. In an effort to study
vascular permeability, we carried out a modified Miles assay in
Cao et al.
Fig. 4. Evans blue was injected intravenously into the tail veins of BALByc mice. After 5 min, 50 ng of leptin or VEGF was administered intradermally in 20 ml
of PBS. The same amounts of FGF-2 and BSA were used as negative controls. The extravasation of Evans blue was recorded with a digital camera system at various
time points (min).
mice. Leptin and VEGF, but not FGF-2 or BSA (data not
shown), induced a rapid vascular permeability response as
measured by the extravasation of Evans blue dye. The leptininduced permeability was detectable 3 min after injection and
reached a maximum after '60 min (Fig. 4). This effect was
almost comparable to that induced by VEGF. Our data show
that leptin acts as a potent vascular permeability factor.
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MEDICAL SCIENCES
Synergistic Angiogenesis. Leptin has been found to be coexpressed
with other angiogenic factors (including FGF-2 and VEGF) in
adipose, placental, and fetal tissues, suggesting that leptin might
modulate the angiogenic activity of these factors (3, 7, 11). To
test this possibility, low doses of leptin and FGF-2 or VEGF were
coimplanted into mouse corneas. At 80 ng per pellet, leptin alone
displayed only a weak stimulation of corneal angiogenesis (Fig.
5e) as measured by vessel density (Fig. 5 j–l) and area (Fig. 5 m
and n). FGF-2 (Fig. 5a) and VEGF (Fig. 5c) at a similar low dose
induced a more intense corneal neovascularization (Fig. 5 f, h,
and k–n). However, a remarkable synergistic effect on the
stimulation of corneal blood vessel growth was observed when
leptin was implanted together with FGF-2 (Fig. 5b) or VEGF
(Fig. 5d). The vessel length, density, and area were all distinctly
increased in leptin and FGF-2 coimplanted corneas (Fig. 5 b, g,
k, and m) or leptin and VEGF coimplanted corneas (Fig. 5 d, i,
l, and n), as compared with those stimulated by leptin alone (Fig.
5 e and j–n), FGF-2 alone (Fig. 5 a, f, k, and m), or VEGF alone
Fig. 5. (a–e) Approximately 80 ng of leptin (e), 40 ng of FGF-2 (a), 80 ng of VEGF (c), and the same amounts of leptin plus FGF-2 (b) and leptin plus VEGF (d)
were implanted into each corneal micropocket of mice. Corneas were photographed by a stereomicroscope on day 5 after growth factor implantation; positions
of implanted pellets are indicated by white arrows in a–e. The area of neovascularization (m and n) was calculated as described (28, 29) and presented as mean
determinants (6SEM) of 10 –12 corneas in each group. ( f–j) Corneal sections were incubated with an anti-CD31 antibody and stained with an FITC-conjugated
secondary antibody. Immunohistological sections of the leptin-implanted cornea (j), FGF-2-implanted cornea ( f), VEGF-implanted cornea (h), leptin plus
FGF-2-implanted cornea (g), and leptin plus VEGF-implanted cornea (i). Corneal microvessels are revealed in green color. Vessel counts (k and l) are presented
as mean determinants (6SEM) of 6 – 8 corneas in each group. *, P , 0.05; **, P , 0.01; ***, P , 0.001.
Cao et al.
PNAS u May 22, 2001 u vol. 98 u no. 11 u 6393
(Fig. 5 c, h, l, and n). The measured area and density of
neovascularization induced by leptin and FGF-2, or leptin and
VEGF, were statistically greater than the sum of the effects
obtained with these factors used alone. Thus, leptin stimulates
angiogenesis synergistically with FGF-2 and VEGF. The calculated P values for vessel area were P , 0.001 in FGF-2 and leptin
coimplanted corneas vs. the sum of FGF-2 alone plus leptin
alone, and P , 0.01 in VEGF and leptin coimplanted corneas vs.
the sum of VEGF alone plus leptin alone. The calculated P
values for vessel density were P , 0.05 in FGF-2 and leptin
coimplanted corneas vs. the sum of FGF-2 alone plus leptin
alone, and P , 0.01 in VEGF and leptin coimplanted corneas vs.
the sum of VEGF alone plus leptin alone.
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Discussion
The angiogenic function of leptin demonstrates that this endocrine hormone acts as a paracrine growth factor for the vascular
system. By stimulating local angiogenesis, leptin may promote its
own release into the circulatory system to regulate the hypothalamus-mediated satiety effect that partially contributes to the
maintenance of body-weight homeostasis (12). In addition,
leptin-induced angiogenesis may increase fatty acid oxidation
and maintain an appropriate balance between blood supply and
fat deposits in adipose tissues. The maintenance of such a
balance requires the exchange of molecules between the blood
stream and adipose tissues. Adipose tissues, like most other
endocrine organs, have to secrete hormones into the blood
stream. The release of hormones into the circulatory system may
require hyperpermeable vessels that may contain fenestrated
capillaries. The different responses of corneal angiogenesis
induced by leptin, FGF-2, and VEGF imply that different,
although overlapping, signaling pathways in endothelial cells
could be involved in the angiogenic responses stimulated by
these factors. The fact that vascular fenestrations are present in
both leptin- and VEGF-induced, but not in FGF-2-induced new
blood vessels, suggests that leptin and VEGF could be involved
in the maintenance of fenestrated endothelium in adipose tissue
and endocrine organs. VEGF was discovered as a potent vascular
permeability factor, and its permeability action has been correlated with the increase of endothelial fenestrations (5, 13). The
energy expenditure and hormone release of leptin in adipose
tissues require permeable blood vessels. Thus, vascular permeability induced by leptin in tissues might provide a mechanism by
which the excess amounts of leptin would be exported into the
circulation. Thus, the regulation of vascular permeability could
act as a gatekeeper to control the actions of leptin as an
endocrine hormone or as an angiogenic factor.
The leptin receptor consists of five spliced isoforms (14). The
longest form, Ob-Rb, contains an active signaling site in the
cytoplasmic domain. In dbydb mice, a premature stop codon is
inserted into the cytoplasmic region, resulting in obesity. The
Ob-Rb is primarily expressed in neuronal tissue (15). Other
spliced short forms of leptin receptors are expressed in many
tissues. It is not known which isoform of receptors mediates
vascular permeability. However, according to previous receptor
studies, certain short isoforms of receptors (including Ob-Ra)
could mediate biological signals (16). Thus, it is possible that the
short form of leptin receptor could mediate vascular permeability. This hypothesis is supported by the fact that high levels of
leptin have been detected in the plasma of dbydb mice as
compared with wild-type mice (17). This observation supports
our hypothesis that leptin could act as a gatekeeper for its own
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6394 u www.pnas.orgycgiydoiy10.1073ypnas.101564798
release. In other words, the more leptin that is synthesized, the
greater is the amount released into the circulation.
The molecular mechanisms of the control of vascular fenestrations are poorly understood. Interestingly, VEGF is also
produced by adipocytes (18). Thus, the regulation of vascular
fenestrations in adipose tissue seems to be complex and involves
several factors. The presence of capillary fenestrations in adipose
tissue in normal mice, but not in obyob mice, suggests that leptin,
but not VEGF, is the key molecule in the maintenance of
capillary fenestrations in the adipose tissue.
Another site (in addition to adipose tissue) where leptin is
produced at a high level is the placenta, a highly angiogenic tissue
(19). The biological function of leptin in the placenta is not known,
but our data suggest that in addition to its angiogenic effect, leptin
may increase the exchange of small molecules between the maternal
circulation and the fetus by the induction and maintenance of
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It seems that leptin may not be the only endocrine hormone
that stimulates angiogenesis and regulates vascular permeability.
For example, the growth hormone family has been found to have
angiogenic properties (20, 21). Corticotropin-releasing hormone
(CRH) has been reported to act as a proangiogenic molecule
(22). In contrast to leptin, CRH reduces vascular permeability
(22–24). In addition, gonadotropin, another endocrine hormone,
also reduces vascular permeability (25). Thus, it is possible that
vascular permeability in an organ represents the balanced status
controlled by both vascular permeability enhancing and inhibitory factors.
In this paper, we also provide evidence that leptin modulates
angiogenic responses induced by FGF-2 and VEGF. Leptin and
FGF-2 or VEGF could produce synergistic effects in stimulation
of blood vessel growth in tissues where these factors are coexpressed. For example, all three of these factors have been found
to be expressed at high levels in the developing placenta and in
several regions of the fetus, including heart and bone (3). The
signaling pathways stimulated by leptin in endothelial cells
support its angiogenic synergism with FGF-2 and VEGF. Leptinstimulated angiogenesis is mediated through the Jak-STAT
(Janus-tyrosine kinase–signal transducers and activators of transcription) pathway, whereas VEGF-induced angiogenesis is most
likely mediated by the protein kinase C pathway, and FGF-2induced angiogenic response is transduced by mitogen-activated
protein kinases-regulated pathways (1, 26, 27).
We show in this paper that leptin-induced new blood vessels
are fenestrated and permeable, and that leptin displays a synergistic effect with FGF-2 or VEGF in the stimulation of
blood-vessel growth. Our data demonstrate that several angiogenic factors are more effective than a single molecule in
stimulation of angiogenesis.
We thank Duojia Cao for her valuable technical assistance and for discussions. We thank Niina Veitonmaki and Anna Eriksson for reading the
manuscript and for discussions. This work was supported by the Swedish
Cancer Foundation Grant 3811-B00–05XAC (to Y.C.), by a grant from the
Human Frontier Science Program (to Y.C.), by the Karolinska Institute
Foundation, the Magnus Bergvalls Foundation, the Ake Wibergs Foundation, Pharmacia, and Upjohn. R.C. is supported by Pharmacia and Upjohn
and the David and Astrid Hagelens Foundation. J.T. is supported by Grant
06537 from the Swedish Medical Research Council, by the Swedish Heart
Lung Foundation, and by the King Gustaf V 80th Birthday Fund.
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