Am J Physiol Endocrinol Metab 295: E313–E322, 2008.
First published May 20, 2008; doi:10.1152/ajpendo.90296.2008.
Macrophage infiltration into adipose tissue may promote angiogenesis
for adipose tissue remodeling in obesity
Can Pang,1 Zhanguo Gao,2 Jun Yin,2 Jin Zhang,2 Weiping Jia,1 and Jianping Ye2
1
Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai
Diabetes Institute, Shanghai Clinical Center of Diabetes, Shanghai, China; and 2Pennington Biomedical Research Center,
Louisiana State University, Baton Rouge, Louisiana
Submitted 14 March 2008; accepted in final form 13 May 2008
Pang C, Gao Z, Yin J, Zhang J, Jia W, Ye J. Macrophage
infiltration into adipose tissue may promote angiogenesis for adi-
pose tissue remodeling in obesity. Am J Physiol Endocrinol Metab
295: E313–E322, 2008. First published May 20, 2008;
doi:10.1152/ajpendo.90296.2008.—The biological role of macro-
phage infiltration into adipose tissue in obesity remains to be fully
understood. We hypothesize that macrophages may act to stimulate
angiogenesis in the adipose tissue. This possibility was examined by
determining macrophage expression of angiogenic factor PDGF
(platelet-derived growth factor) and regulation of tube formation of
endothelial cells by PDGF. The data suggest that endothelial cell
density was reduced in the adipose tissue of ob/ob mice. Expression
of endothelial marker CD31 was decreased in protein and mRNA. The
reduction was associated with an increase in macrophage infiltration.
In the obese mice, PDGF concentration was elevated in the plasma,
and its mRNA expression was increased in adipose tissue. Macro-
phages were found to be a major source of PDGF in adipose tissue, as
deletion of macrophages led to a significant reduction in PDGF
mRNA. In cell culture, PDGF expression was induced by hypoxia,
and tube formation of endothelial cells was induced by PDGF. The
PDGF activity was dependent on S6K, as inhibition of S6K in
endothelial cells led to inhibition of the PDGF activity. We conclude
that, in response to the reduced vascular density, macrophages may
express PDGF in adipose tissue to facilitate capillary formation in
obesity. Although the PDGF level is elevated in adipose tissue, its
activity in angiogenesis is dependent on the availability of sufficient
endothelial cells. The study suggests a new function of macrophages
in the adipose tissue in obesity.
macrophage; platelet-derived growth factor; angiogenesis; ribosomal
protein S6 kinase; hypoxia; obesity
TISSUE REMODELING IS INVOLVED in the expansion of adipose
tissue during body weight gain. Angiogenesis is a critical event
in tissue remodeling. Angiogenesis may be coupled with adi-
pogenesis during adipose tissue remodeling throughout life-
time (11, 28, 33, 34). The increase in adipocyte size is asso-
ciated with compensation in the microcirculation, as has been
reviewed (5, 11). Inhibition of angiogenesis prevents fat mass
expansion in several rodent models of obesity (6, 41). In
addition to the well-known adipokines (including adiponectin,
leptin, TNF-␣, and resistin), adipose tissue also produces a
variety of endothelial cell growth factors and angiogenic fac-
tors (11), such as fibroblast growth factor (FGF), vascular
endothelial growth factor (VEGF), platelet-derived endothelial
Address for reprint requests and other correspondence: J. Ye, Pennington
Biomedical Research Center, Louisiana State University, Baton Rouge, LA
70808 (e-mail: yej@pbrc.edu) or W. Jia, Dept. of Endocrinology and Metab-
olism, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shang-
hai Diabetes Institute, Shanghai Clinical Center of Diabetes, Shanghai, China
(e-mail: wpjia@yahoo.com).
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cell growth factor (PD-ECGF), transforming growth factor
(TGF)-␤, and angiopoietin. The functions of these factors are
demonstrated in experimental angiogenesis assays with condi-
tioned media obtained from preadipocytes and tissue homog-
enates of fat tissues (8, 17, 44). The factor that induces
expression of the angiogenic factors or vascular compensation
remains to be identified in adipose tissue. In a recent study, we
demonstrated hypoxia in the adipose tissue in ob/ob and dietary
obese mice (56). Adipose tissue hypoxia may play a role in the
induction of these angiogenic factors. Cross-talk between adi-
pocytes and endothelial cells has been supported by many
studies and is required for adipose tissue growth (8, 27, 34, 50).
However, not much is known about cross-talk between mac-
rophages and endothelial cells in adipose tissue in tissue
growth or remodeling.
Macrophage infiltration into adipose tissue contributes to the
increased expression of inflammatory cytokines in obesity (52,
55). It remains to be investigated why macrophage infiltration
is increased in adipose tissue. Some studies suggest that mac-
rophage infiltration is for clearance of dead adipocytes in the
adipose tissue (10, 45). Except for chronic inflammation and
clearance of dead cells, the biological significance of macro-
phage infiltration remains largely unknown in the adipose
tissue. In wound healing and tumor growth, macrophage infil-
tration contributes to the stimulation of angiogenesis (46).
Such a role of macrophages remains to be established in
adipose tissue remodeling in obesity. We hypothesized that
macrophage infiltration might be involved in the stimulation of
angiogenesis during expansion of adipose tissue.
As a proangiogenic factor in serum, platelet-derived growth
factor (PDGF) stimulates differentiation of endothelial cells
and migration of pericytes (21, 22, 29, 40). Although PDGF is
secreted by many types of cells, including platelets, macro-
phages, fibroblasts, and endothelial cells, macrophages are one
of the major sources of PDGF in tissues (32, 43). Among the
three active isoforms of PDGF (AA, AB, and BB), PDGF-BB
is able to activate all of the three PDGF receptors (␣␣, ␣␤, and
␤␤), which are dimeric tyrosine kinases. The importance of
PDGF in the regulation of vascular development and function
was demonstrated in gene knockout mice in which inactivation
of either PDGF-B or its receptor was embryonically lethal (29).
The mice died from hemorrhage, edema, and absence of kidney
glomerular mesangial cells. In addition to the regulation of
vascular development, PDGF is also involved in oncogenesis,
atherosclerosis, lung fibrosis, kidney fibrosis, etc. (20). Al-
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E314 REGULATION OF ADIPOSE TISSUE ANGIOGENESIS BY MACROPHAGES
though PDGF expression is elevated locally in tissue remod-
eling processes such as wound healing and tumor growth, it is
not clear whether PDGF expression is increased during adipose
tissue growth in obesity. As hypoxia induces PDGF expression
(16), we propose that PDGF expression may be increased by
adipose tissue hypoxia and may be involved in stimulation of
angiogenesis during adipose tissue remodeling in obesity.
The angiogenic activities of PDGF are mediated by signal-
ing pathways of cell membrane receptors of PDGF (47).
Ligand engagement leads to PDGF receptor phosphorylation
and activation of several signaling pathways, including Src,
PI3K, and phospholipase C␥ (PLC␥) (47). The phophatidyli-
nositol 3-kinase (PI3K)/Akt/mTOR (mammalian target of
rapamycin) pathway is activated by PDGF (25, 58) and is
involved in the recruitment of pericytes (13). It remains to be
tested whether the PI3K/Akt/mTOR pathway is involved in
tube formation of endothelial cells in response to PDGF. In
studies of other angiogenic factors, the PI3K/Akt/mTOR path-
way was reported to stimulate proliferation (51) and tube
formation of vascular endothelial cells (57). However, there is
no direct evidence that this signaling pathway is required by
PDGF in the stimulation of tube formation.
In this study, we demonstrated that vascular density was
reduced in adipose tissue in ob/ob mice. PDGF expression was
elevated in adipose tissue and expressed in macrophages. In
cell culture, PDGF stimulated tube formation of endothelial
cells, and the activity required ribosomal protein S6 kinase
(S6K). These data suggest that macrophage PDGF may play an
important role in the stimulation of tube formation in the
process of angiogenesis in adipose tissue. The study provides
direct evidence for the role of the PI3K/mTOR/S6K signaling
pathway in the PDGF-induced tube formation.
EXPERIMENT PROCEDURES
Animals. Male C57BL/6J-Lepob, and C57BL/6J mice were pur-
chased from the Jackson Laboratory (Bar Harbor, ME) at 6 wk of age
and used in the study according to the animal protocol approved by
the Institutional Animal Care and Use Committee at the Pennington
Biomedical Research Center, Louisiana State university (Baton
Rouge, LA). The mice were housed in regular cage with four mice per
cage with free access to water and standard chow unless noted
otherwise. The serum of the mice was collected from the tail vein. The
serum level of PDGF was quantified with a Mouse/Rat PDGF-BB
Immunoassay kit (MBB00; R&D Systems).
Cell lines and reagents. Murine endothelial cell line SVEC4-10
(CRL-2181, ATCC), murine fibroblast cell line 3T3-L1 (CL-173,
ATCC), and the RAW 264.7 cell line (TIB-71) were maintained in
DMEM with 10% fetal bovine serum in a CO2 (5%) incubator.
3T3-L1 preadipocytes were differentiated into adipocytes as described
elsewhere (15). Antibodies to phospho-p70 S6 kinase (Thr421/Ser424,
9204) and phospho-Akt (Ser473, 9271) were obtained from Cell
Signaling (Boston, MA). Antibodies to PDGF-BB (ab53716), phospho-
glycogen synthase kinase (GSK)-3␤ (Ser9, ab30619), GSK-3␤
(ab31366), S6K (ab9366), tubulin (ab7291), and actin (ab8227) were
obtained from Abcam (Cambridge, MA). Antibodies to HA (sc-7392)
and Akt1 (sc-8312) were bought from Santa Cruz Biotechnoloty (Santa
Cruz, CA). Rapamycin (A275-0001), LY-294002 (ST-420), and SP-
600125 (EI-305) were obtained from Biomol International (Plymouth
Meeting, MA). Wortmannin (W1628) and SB-203580 (S8307) were
obtained from Sigma-Aldrich (St. Louis, MO). These reagents were
diluted and added to the cells at indicated working concentrations.
Deletion of macrophages in adipose tissue. Macrophages were
deleted in the adipose tissue by a single injection of clodronate
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liposome. Clodronate liposome was prepared and administration at
100 –150 mg/kg ip as described elsewhere (49). The macrophage
deletion was confirmed in adipose tissue at day 4 after injection.
Tube formation assay. Tube formation assay was conducted on the
Matrigel (35027, BD Biosciences), which was added in a volume of
50 ␮l/well to a 96-well plate (3585, Fisher Scientific) and allowed to
polymerize at 37°C for 30 min. After polymerization, the endothelial
cells were plated on the Matrigel at 2 ⫻ 104 cells/well in 200 ␮l of
serum-free medium with or without reagents. The cells were incu-
bated at 37°C with 95% humidity and 5% CO2. The tube formation
was observed under an inverted microscope (Zeiss Axiovert 40 CFL)
after 10 h. Images were captured with a Zeiss AxioCam Hrc CCD
camera attached to the microscope. The tube formation was quantified
by measuring the long axis of the individual cells on Matrigel using
NIH Image J (version 1.31). A mean value of total length in each
sample was used to represent the tube formation.
Protein extract and Western blot. SVEC4-10 cells (5 ⫻ 105/well)
were plated in a 12-well plate in DMEM supplemented with 10% FBS
for 24 h. Then the cells were starved overnight in serum-free medium
and treated with various reagents for 30 min. Protein extraction and
Western blot analysis were conducted for analysis of signaling activ-
ities in cells, as described elsewhere (15). The intensity of Western
blot signal was quantified with NIH Image J, and the signal was
normalized against loading control.
Generation of stable cell line with dominant-negative S6K mutant.
The SVEC4-10 endothelial cells were cotransfected with 3 ␮g of
pRK7-HA-S6K1-KR plasmid for dominant-negative S6K (8985, Ad-
dgene) and 0.5 ␮g of pcDNA3.1 plasmid for neomycin resistance. In
the control, the cells were transfected with pcDNA 3.1 plasmid. The
transfection was conducted with Lipofectamine 2000 in 4.5 ml of
medium. The transfected cells were cultured in G418 (300 mg/ml)-
containing medium 36 h later for 24 –28 days. The stable positive cells
were confirmed in Western blot with HA antibody and S6K rabbit
antibody. The positive cells were cultured in G418-free DMEM for
two passages before experiment.
Cell viability (MTT) assay. SVEC4-10 (1 ⫻ 104 cells/well) were
plated in a 96-well plate in DMEM supplemented with 10% FBS.
Then the cells were starved overnight in a serum-free medium and
treated with various reagents for 24 h. The MTT assay was conducted
by incubation of the cells in 20 ␮l of MTT solution (5 mg/ml in PBS)
for 3–5 h. Cell viability was determined by formazan (MTT metabolic
product), which was quantified with optical density at 560 nm in 200
␮l of DMSO and normalized over the subtract background at 670 nm.
Hypoxia treatment. Cells were treated with hypoxia (1% oxygen)
in vitro, as described elsewhere (56). The control cells were main-
tained in normoxic condition. After treatment, the cell culture super-
natant was collected as conditioned medium and stored at ⫺80°C until
experiments (30).
Quantitative RT-PCR. Total RNA was extracted from homoge-
nized fat pads or cells using Tri Reagent (T9424; Sigma, St. Louis,
MO). Quantitative real-time RT-PCR (qRT-PCR) was conducted
using the ABI 7900HT fast real-time PCR system (Applied Biosys-
tems, Foster City, CA). The following primers and probes were
ordered from Applied Biosystems: PDGF (Mm00437304_m1), CD31
(Mm01246167_m1), and F4/80 (Mm00802530_m1). The specific
signal was normalized over 18S rRNA. A mean value of triplicates
was used to express gene expression.
Immunohistochemistry. The epididymal fat pads were isolated,
fixed in neutral buffered formalin, dehydrated, and embedded in
paraffin. Thin tissue slides (5 ␮m) were deparaffinized, blocked, and
incubated overnight at 4°C with a mouse anti-mouse CD31 antibody
(ab24590; Abcam, Cambridge, MA), which was followed by signal
amplification using a VECTASTAIN Elite ABC Kit (PK-6102; Vec-
tor Laboratories). The reaction was developed by addition of AEC
chromogen substrate (AEC Staining Kit; Sigma-Aldrich). In fluores-
cence analysis, the CD31 signal was determined with goat anti-mouse
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 REGULATION OF ADIPOSE TISSUE ANGIOGENESIS BY MACROPHAGES
IgG-FITC (sc-2010, Santa Cruz Biotechnology). Microphotographs
were taken under a microscope (⫻20).
Peritoneal macrophages. Primary peritoneal macrophages were
isolated from C57BL/6J mice, as described elsewhere (56). The
macrophages were transferred to 35-mm tissue culture dishes and
treated with hypoxia in serum-free medium 3 days later for collection
of supernatant.
Statistical analysis. All of the experiments were conducted at least
three times with consistent results. The gel or image from represen-
tative experiment is presented. Values are means ⫾ SE of multiple
data points. Student’s t-test or one-way ANOVA was used in statis-
tical analysis of the data with a significance of P ⬍ 0.05.
RESULTS
Decreased density of microvasculature in adipose tissue in
ob/ob mice. A decrease in capillary density has been proposed
as a mechanism of blood flow reduction in adipose tissue in
obesity. However, the reduction in capillary density is contro-
versial (7, 23). One possible reason for the discrepancy is the
quantification method for capillary density, which was deter-
mined by immunostaining in the studies (7, 23). To resolve the
discrepancy, we compared capillary density in adipose tissues
of lean mice and ob/ob mice via quantification of endothelial
cell marker CD31. The CD31 protein was examined by immu-
nostaining and Western blot. The CD31 mRNA was determined
by qRT-PCR. In the immunohistostaining, the CD31 protein was
detected in the tissue with either colorimetric- or fluorescence-
based staining. CD31, distributed in the extracellular matrix of
adipocytes, was reduced in obese mice (Fig. 1, A and B). The
protein and mRNA of CD31 were both reduced in ob/ob mice
(Fig. 1, C and D). These data suggest that neovascularization is
reduced in white adipose tissue in obesity.
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PDGF expression in adipose tissue of ob/ob mice. To
understand the reduction in capillary density, we examined
PDGF activity in the adipose tissue of obese mice. As a
proangiogenic factor, PDGF is increased in human serum in
response to inflammation and is involved in angiogenesis in
many tissues (1, 3). However, it is not clear whether blood
PDGF is elevated in an obese condition. To address this issue,
the PDGF protein was compared in the serum of lean and ob/ob
mice. The PDGF protein was increased in obese mice, as
indicated by the ELISA data (Fig. 2A). To determine the source
of PDGF protein, PDGF mRNA was determined in the adipose
tissue by qRT-PCR. The mRNA was increased in the ob/ob
mice as well (Fig. 2B). To determine the cell types for the
source of increased PDGF, we examined PDGF mRNA in the
adipose tissue after deletion of macrophages in the obese mice.
The PDGF expression was reduced by macrophage deletion in
a dose-dependent manner (Fig. 2C). These data suggest that
PDGF is elevated in the blood and that adipose tissue may
contribute to the systemic increase in PDGF protein. Macro-
phages are responsible for the PDGF increase in adipose tissue.
Expression of PDGF by macrophages and adipocytes. The
data above suggest that macrophages may be a major producer
of PDGF in the adipose tissue of obese mice. To test this
question, we compared macrophages and adipocytes for ex-
pression of PDGF mRNA. The primary cells and cell lines
were used and their activities compared in the basal and
hypoxia-treated conditions. In the primary cells, macrophages
expressed more PDGF than the adipocytes in the basal condi-
tion (Fig. 3A). This pattern of expression was also observed in
the cell lines of macrophages (RAW cell) and adipocytes
(3T3-L1) (Fig. 3B). These data suggest that PDGF is expressed
Fig. 1. Vascular density decreased in adipose
tissue in ob/ob mice. A and B: sections of
epididymal fat pads were stained with CD31
antibody to reveal endothelial cell density.
Immunohistostaining was conducted for the
endothelial marker CD31. A: CD31 protein is
indicated in red from AEC staining. B: CD31
protein is indicated in green fluorescent. Blue
stands for nuclei stained by DAPI. Scale bar,
50 ␮m. C: CD31 protein was determined in
whole cell lysate of epididymal fat pads in
Western blot. Representative blot is shown
with an average signal strength in the bar
figure. D: CD31 mRNA was determined in
epididymal fat pads by qRT-PCR. In this
figure, each data point represents mean ⫾ SE
(n ⫽ 5). Experiments were repeated 3 times
with consistent results.
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E316 REGULATION OF ADIPOSE TISSUE ANGIOGENESIS BY MACROPHAGES

Fig. 2. Platelet-derived growth factor (PDGF) increased in adipose tissue in ob/ob mice. A: serum PDGF was determined by ELISA. B: PDGF mRNA was
determined in epididymal fat pads by qRT-PCR. C: PDGF mRNA in adipose tissue after macrophage deletion. Macrophages were reduced in adipose tissue by
single ip injection of clodronate liposome in ob/ob mice (6 wk old). PDGF was examined at day 4 after injection. F4/80 is a marker of macrophage. Each data
points represents mean ⫾ SE (n ⫽ 3).

in adipocytes, but the expression level is significantly lower
than that of macrophages. The impact of differentiation on
PDGF expression was investigated in 3T3-L1 cells. After
differentiation, PDGF expression was significantly reduced in
3T3-L1 cells (Fig. 3C). These data suggest that expression of
PDGF is in the order of macrophage ⬎ preadipocytes ⬎
mature adipocytes.
Induction of PDGF expression by hypoxia. Hypoxia in
adipose tissue may induce PDGF expression in obese mice, as
PDGF-B is a hypoxia response gene (16); this possibility
remained to be tested. To test this, PDGF expression was
determined in primary macrophages after exposure to ambient
hypoxia. As expected, PDGF expression was increased by the
hypoxia treatment (Fig. 3D). In the macrophage cell line
(RAW), the same effect was observed for hypoxia (Fig. 3D).
However, the response of RAW macrophages was stronger
than for the primary macrophages. These data further support
that macrophages are a the major producer of PDGF in the
adipose tissue of obese mice. Macrophage infiltration should
contribute to the elevated PDGF expression in adipose tissue.

Fig. 3. PDGF expression in macrophages and adipocytes. A: PDGF mRNA in primary cells. The basal level of PDGF mRNA was determined in primary
macrophages and adipocytes of lean mice. B: basal level of PDGF mRNA was determined in macrophage cell lines (RAW cells) and 3T3-L1 adipocytes. C: basal
level of PDGF mRNA was determined in 3T3-L1 fibroblasts before and after differentiation into adipocytes. D: induction of PDGF mRNA expression by ambient
hypoxia in primary macrophages and RAW macrophages. E: expression of PDGF in adipose tissue of lean mice after macrophage deletion. A single injection
of clodronate liposome (150 mg/kg) was used to delete macrophages in lean C57BL/6J mice (8 wk old). Experiments were repeated 3 times with consistent
results. Each data point represents mean ⫾ SE (n ⫽ 3). *P ⬍ 0.05, treated vs. untreated in RAW cells; ⫹P ⬍ 0.05, treated vs. untreated in primary macrophages.

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 REGULATION OF ADIPOSE TISSUE ANGIOGENESIS BY MACROPHAGES
In lean mice, preadipocytes or stromal cells may be the primary
source of PDGF, as deletion of macrophages did not lead to
reduction in PDGF expression (Fig. 3E).
Function of PDGF in regulation of angiogenesis. Given the
role of PDGF in angiogenesis, we proposed that macrophage
PDGF may be involved in the stimulation of capillary growth
in adipose tissue under hypoxia, in which case the supernatant
of hypoxia-treated macrophages should be able to stimulate
angiogenesis. To test this possibility, the supernatant of RAW
macrophages treated by hypoxia was examined in the tube
formation assay. Such an assay was used to study PDGF
activity elsewhere (2, 12). The results suggest that the macro-
phage supernatant induced more tube in the assay, and this
activity increased in the supernatant of hypoxia-treated mac-
rophages (Fig. 4, A and B, control antibody). In the presence of
PDGF antibody, such an increase in tube formation was inhib-
ited (Fig. 4, A and B, PDGF antibody).
PI3K/Akt pathway in PDGF-stimulated tube formation. The
signaling pathway of PDGF receptor is involved in angiogen-
esis and is described in several pathological conditions (47).
The importance of the PI3K/Akt/mTOR pathway in PDGF-
induced angiogenesis was demonstrated in vivo in rodent
models (13, 18, 25, 58). PDGF is known to activate the
Fig. 4. Induction of tube formation by PDGF in macrophage supernatant. Tube
formation of endothelial cells on Matrigel was used to test proangiogenic
activity of PDGF in supernatants of macrophages. Raw cells were treated with
hypoxia for 4 h, and its supernatant was collected for induction of tube
formation in endothelial cells. PDGF antibody was used to determine activity
of PDGF in the supernatant. A: representative images of tube formation of the
endothelial cells on Matrigel. B: quantification of tube formation. Experiments
were repeated 3 times with consistent results. Each data point represents
mean ⫾ SE (n ⫽ 3 experiments).
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E317
PI3K/Akt pathway in pericytes that normally form part of the
capillary wall. It remains to be tested whether the same path-
way is involved in tube formation by endothelial cells. To
investigate the PI3K/Akt pathway in endothelial cells, we exam-
ined PI3K inhibitors on tube formation. In the positive control,
insulin was used to induce tube formation (Fig. 5, A and B). In the
presence of PI3K inhibitor LY-294002 (100 ␮M) or wortmanin
(1 ␮M), the insulin-induced tube formation was reduced sig-
nificantly. Recombinant PDGF was used to stimulate tube
formation in the mouse endothelial cells. As expected, the tube
formation was induced by the recombinant PDGF, and the
effect was blocked by the PI3K inhibitors (Fig. 5, A and B). The data
suggest that PI3K activity is required for tube formation
induced by PDGF and insulin. The basal level of tube forma-
tion was also reduced by the PI3K inhibitors (Fig. 5, A and B),
suggesting that PI3K activity is required for tube formation
induced by Matrigel, which contains proangiogenic factors.
To verify activation of PI3K/Akt by PDGF, we examined
the phosphorylation status of Akt and its downstream mole-
cules in a Western blot. Identical to those observed with insulin
in the positive control, the activation markers of Akt, S6K, and
GSK3-␤ were all increased by PDGF (Fig. 5, C and D). These
changes were consistently blocked by the PI3K inhibitor LY-
294002. We also tested the roles of MAPK kinases in PDGF-
induced tube formation with chemical inhibitors to ERK (PD-
098095 40 ␮M), JNK (SP-600125 50 ␮M), and p38 (SB-
203580 2 ␮M). Although these inhibitors could inhibit the
basal level of tube formation, they did not decrease the PDGF-
induced tube formation significantly (Supplemental Fig. S1,
online only). These data suggest that the PI3K/Akt pathway is
required for angiogenesis induced by PDGF.
mTOR in PDGF-induced tube formation. mTOR is a major
signaling molecule downstream of Akt. Inhibition of angiogen-
esis by rapamycin in mice suggests that mTOR may mediate
PI3K/Akt signaling in the stimulation of angiogenesis in mice
(18). In the current study, inhibition of mTOR led to a reduc-
tion in tube formation induced by PDGF as well as by insulin
(Fig. 6, A and B), suggesting that mTOR is required for the
PDGF signaling pathway. Activation of mTOR by PDGF and
its inhibition by rapamycin were examined by the phosphory-
lation status of S6K in Western blot. As was observed for
insulin, the phosphorylation was induced by PDGF and
blocked by rapamycin (Fig. 6, C and D). As inhibition of
protein synthesis by cycloheximide abolished the tube forma-
tion induced by PDGF or insulin (Fig. 6A), protein synthesis is
required for the tube formation. This observation suggests that
mTOR may act through S6K in the PDGF signaling pathway in
endothelial cells, as S6K controls gene translation.
S6K in PDGF-induced tube formation. S6K is one of the
major signaling molecules activated by mTOR and is involved
in regulation of protein synthesis. It remains to be tested
whether S6K mediates PDGF signal in endothelial cells for
tube formation (24, 38, 57). The S6K dominant-negative (S6K-
DN) mutant in stable transfection was generated to test S6K
activity in PDGF-induced tube formation. The mutant S6K was
expressed in the SVEC4-10 cells, as indicated by detection of
HA-tag in Western blot (Fig. 7A). Inhibition of S6K function
was confirmed, with decreased phosphorylation of its substrate
S6 in the transfected cells (Fig. 7B). In response to PDGF, the
stable cell line expressing S6K-DN exhibited much fewer tubes
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E318 REGULATION OF ADIPOSE TISSUE ANGIOGENESIS BY MACROPHAGES

Fig. 5. PI3K/Akt pathway in PDGF-induced tube formation. The role of PI3K in tube formation induced by PDGF was examined using PI3K inhibitors.
A: representative images of tube formation of the endothelial cells induced by PDGF. B: quantification of tube formation. C: representative Western blots of
phosphorylation of PI3K/Akt signaling molecules. D: quantification of Western blot signals. In this figure, all experiments were repeated 3 times with consistent
results. Each data point represents mean ⫾ SE (n ⫽ 3 experiments).

on the Matrigel (Fig. 7, C and D), suggesting that S6K is
required by PDGF in the signal transduction.
Stimulation of tube formation by albumin. The data above
suggest that the signal of tube formation is transduced through
PI3K/Akt/mTOR/S6K under the PDGF receptor. It is not clear
whether activation of S6K in the absence of activation of
upstream signaling molecules including PI3K and Akt is suf-
ficient to induce tube formation. To test this possibility, S6K
was activated with albumin (Fig. 8A). The activation led to an
increase in tube formation, and the increase was blocked by
rapamycin (Fig. 8, B and C), which suppresses S6K by target-
ing mTOR. The data suggest that activation of S6K by albumin
is able to increase tube formation. Toxicity of rapamycin was
examined in an MTT assay. Treatment of the cells with
rapamycin for 24 h had no significant effect on cell viability in
presence or absence of PDGF (Fig. 8D).
DISCUSSION
Our study suggests that vascular density is decreased in
adipose tissue in ob/ob mice. Angiogenesis is required for the
development and growth of adipose tissue (7, 41). During
development of obesity, angiogenic activity will be increased
to compensate for expansion of adipocyte tissue. This compen-
sation ensures quick growth in adipose tissue at the early stage
of weight gain. Once the weight gain reaches a level such as
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BMI ⬎25–30 kg/m2, a failure in angiogenic compensation may
occur and thus reduce the growth rate of fat tissues. A reduc-
tion in adipose tissue blood flow may be a result of such
angiogenic failure (53). To explain adipose tissue hypoxia in
the obese condition (56), we proposed that angiogenic failure
may occur in adipose tissue of obese mice. In the current study,
this possibility is supported by the reduction of vascular den-
sity in the adipose tissue. The CD31 data from immunohis-
tostaining, Western blot, and qRT-PCR consistently support
the lack of endothelial cells in the adipose tissue of ob/ob mice
(Fig. 1). A lack of VEGF induction by hypoxia was observed
in the adipose tissue in the same condition (56). The VEGF
non-response may contribute to the reduced endothelial cells,
as VEGF is a primary growth factor for endothelia cells.
Macrophages may serve as a stimulator for angiogenesis in
adipose tissue in obesity. Adipose tissue contains several types
of cells, such as preadipocytes (or stromal cells), adipocytes,
macrophages, and endothelial cells. PDGF is expressed in all
of these types of cells; however, the expression levels are
different. The current study suggests that preadipocytes ex-
press more PDGF than mature adipocytes. In obesity, preadi-
pocyte number is reduced, as most of them are differentiated
into mature adipocytes. This change may lead to a decrease in
local PDGF level, since mature adipocytes produce less PDGF
than the preadipocytes (Fig. 3C). To meet the demand for
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 REGULATION OF ADIPOSE TISSUE ANGIOGENESIS BY MACROPHAGES E319

Fig. 6. Mammalian target of rapamycin (mTOR) in PDGF-induced tube formation. The role of mTOR/S6K in PDGF-induced tube formation was tested with
mTOR inhibitor (rapamycin) and protein synthesis inhibitor cycloheximide (CHX). A: representative images of tube formation in the presence of rapamycin and
CHX. Insulin was used as a positive control in induction of mTOR activation. Rapamycin (200 nM) and CHX (20 ng/ml) were used to pretreat cells for 30 min,
followed by treatment of PDGF (50 ng/ml) or insulin (100 nM) for 10 h. B: quantification of tube formation. C: representative Western blots for mTOR/S6K
signaling pathway in endothelial cells. Phosphorylation of S6K was determined in SVEC4-10 cells after treatment with PDGF or insulin. D: quantification of
Western blot signals. In this figure, all experiments were repeated 3 times with consistent results. Each data point represents mean ⫾ SE (n ⫽ 3 experiments).

PDGF, macrophage infiltration into adipose tissue is increased
to compensate for the loss of preadipocytes for PDGF produc-
tion (Fig. 2B). Our data support the idea that infiltration and
activation of macrophages may contribute to the increased
PDGF expression in the adipose tissue of obese mice. This
conclusion is supported by the observations that deletion of
macrophages led to a significant reduction in PDGF expression
in adipose tissue of ob/ob mice. The reduction was observed in
obese mice but not in lean mice, which have abundant preadi-
pocytes (Figs. 2C and 3E). Macrophages are second next to
platelets in expression of PDGF (32, 40, 43). We believe that,
in addition to its roles in chronic inflammation (52, 55),
macrophages may serve as an angiogenic stimulator in adipose
tissue by expression of PDGF. To our knowledge, this may be
the first study to support elevation and function of PDGF in
adipose tissue of obesity. Hypoxia in adipose tissue is likely to
induce PDGF expression in macrophages (Fig. 3D). In the
current study, PDGF expression was determined mainly at the
mRNA level. It remains to be tested whether PDGF protein is
induced by hypoxia in macrophages.
We did not find literature about PDGF expression in adipose
tissue in obesity. Closely related literature includes PDGF
regulation of GLUT4 translocation, proliferation, or differen-
tiation of adipocytes in cell culture (19, 31, 36). In vitro, PDGF
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or its receptor has been shown to promote GLUT4 transloca-
tion in adipocytes (31, 36). PDGF has been reported to inhibit
adipocyte differentiation (19). In other organs, local PDGF has
been suggested to play a role in pathogenesis of proliferative
retinopathy and diabetic nephropathy (14, 26, 39). An increase
in PDGF has been found in the retinal membranes and vitreous
fluid of patients with proliferative diabetic retinopathy (14, 39).
A higher PDGF level has also been reported in individuals with
additional rubeosis iridis or ischemic nondiabetic retinopathy
(14). In the renal biopsy of patients with diabetic nephropathy,
expression of PDGF-A and PDGF-B has been reported to be
increased in mRNA and protein (26). In mice with type 1
diabetes, the PDGF elevation has been associated with macro-
phage infiltration into kidney with diabetic nephropathy (9).
These observations suggest a role of PDGF in the pathogenesis
of diabetic complications.
The current study suggests that the activity of PDGF in
angiogenesis is dependent on VEGF activity. Angiogenesis
requires endothelial cell proliferation and tube formation. The
two events are equally important in the process of formation of
capillary. Tube formation requires the presence of endothelial
cells; in the absence of endothelial cells, tube formation will
not occur. Endothelial cell proliferation is dependent on proan-
giogenic factor VEGF, which stimulates cell proliferation
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E320 REGULATION OF ADIPOSE TISSUE ANGIOGENESIS BY MACROPHAGES
Fig. 7. S6K in PDGF-induced tube formation. A: expression of dominant-
negative S6K (S6K-DN) in stable cells. B: inhibition of S6K activity by
S6K-DN mutant. C: tube formation in stable cells in response to PDGF.
D: quantification of tube formation. In this figure, all experiments were
repeated 3 times with consistent results. Each data point represents mean ⫾ SE
(n ⫽ 3 experiments).
through VEGF receptor 2 in the endothelial cells. The lack of
endothelia cells was observed in adipose tissue of ob/ob mice
in this study (Fig. 1). The reduction in endothelial cells is
supported by quantification of CD31 (endothelial cell marker)
in protein and mRNA. This reduction is consistent with our
Fig. 8. S6K in tube formation induced by al-
bumin. A: Induction of phosphorylation of S6K
by albumin. Phosphorylation of S6K was ex-
amined in whole cell lysate in Western blot.
B: BSA-stimulated tube formation and inhibi-
tion by rapamycin (200 nM). C: quantification
of tube formation. D: cell viability after rapa-
mycin treatment. Cell viability was determined
with MTT assay at 24 h after cell exposure to
rapamycin. In this figure, all experiments were
repeated 3 times with consistent results. Each
data points represents mean ⫾ SE (n ⫽ 3
experiments).
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previous observation that VEGF expression was not increased
in adipose tissue by obesity in ob/ob mice (56). Lack of leptin
in the ob/ob mice may be related to the unresponsiveness of
VEGF to obesity or adipose tissue hypoxia. In the wild-type
mice, VEGF was increased by diet-induced obesity in the
adipose tissue (56). Without sufficient endothelial cells, PDGF
will not be able to increase angiogenesis in the adipose tissue
by itself.
In addition to stimulation of tube formation, PDGF was
reported to play a role in the recruitment of pericytes or the
proliferation of vessel smooth muscle cells in angiogenesis.
Recruitment of pericytes is required for maintenance of micro-
vascular stability and function (21, 22, 29). This function of
PDGF is established in a PDGF knockout study (29). Pericytes
are able to transdifferentiate into fibroblasts (stromal cells/
preadipocytes) and vessel smooth muscle cells (37). With these
activities, it is possible that PDGF is involved in recruitment of
preadipocytes into adipose tissue in obesity.
In the current study, we observed that insulin stimulated tube
formation, suggesting a role for insulin in the stimulation of
angiogenesis. This activity indicates that hyperinsulinemia
may serve to stimulate angiogenesis in adipose tissue in the
obese condition. Hyperinsulinemia is a prognosis factor for the
incidence of microvascular abnormalities in diabetes patients
for retinopathy and nephropathy (4). In the present study,
insulin exhibited a similar activity to PDGF in the induction of
tube formation (Figs. 5 and 6). This observation suggests that
hyperinsulinemia may be involved in neovascularation in adi-
pose tissue during the development of obesity.
S6K is required for tube formation induced by PDGF and
insulin. S6K is activated by an array of mitogenic or nutrient
stimuli, such as insulin, serum, phorbol ester, and PDGF (38,
48, 54). Knockout studies suggest that S6K is an important
regulator of cell size and body growth in mice and Drosophila
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 REGULATION OF ADIPOSE TISSUE ANGIOGENESIS BY MACROPHAGES
(35, 42). S6K1⫺/⫺ mice are protected against age- and high-fat
diet-induced obesity and insulin resistance due to increased
energy expenditure (48). In tube formation, S6K may act
through induction of FGF-2 expression in endothelial cells
(57). In the present study, our data suggest that S6K mediates
signals of PDGF and insulin in the induction of tube formation.
This is supported by data that inhibition of S6K by dominant-
negative mutant or chemical inhibitors (LY, wortmanin, and
rapamycin) lead to inhibition of PDGF and insulin activity
(Figs. 5–7). These data consistently support the notion that
S6K is a critical kinase for endothelial differentiation and tube
formation.
In summary, reduction in vascular density is likely a result
of a lack of an endothelium growth factor (such as VEGF) in
adipose tissue in ob/ob mice. This may contribute to the
development of adipose tissue hypoxia and macrophage infil-
tration. PDGF expression is increased in macrophages in re-
sponse to the hypoxia. Although PDGF is able to stimulate
tube formation, it may not stimulate angiogenesis in the ab-
sence of sufficient endothelial cells or VEGF activity. The
signaling pathway PI3K/Akt/mTOR/S6K is used by PDGF in
the induction of tube formation. With insufficient endothelial
cells in the adipose tissue of ob/ob mice, macrophages may fail
to stimulate angiogenesis. These possibilities may support a
new function for macrophages in adipose tissue. Hyperinsulin-
emia may also be involved in angiogenesis in adipose tissue in
obesity by stimulation of tube formation in endothelial cells.
ACKNOWLEDGMENTS
We thank Dr. David Burk in the Imaging Core Facility and Dr. Viktor Drel
for their excellent technical support in the capture of images for tube formation
and processing of tissue slides for immunohistostaining. We thank Hanjie
Zhang for technical support in the preparation of clodronate liposome.
GRANTS
This study is supported by National Institute of Diabetes and Digestive and
Kidney Diseases (NIDDK) funds (DK-68036) and ADA research award
(7-07-RA-189) to J. Ye and the Key Project from the Science and Technology
Commission of Shanghai municipality (04DZ19501, 01ZD002, and National
973 Program) to W. Jia. The qRT-PCR study was conducted in the genomic
core that is supported by the CNRU Grant (1P30 DK-072476) sponsored by
NIDDK.
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