Stem Cells Express, published online September 4, 2008; doi:10.1634/stemcells.2008-0261

STEM CELLS®
TISSUE-SPECIFIC STEM CELLSIFATS Series: FGF-2-induced HGF Secretion By Adipose-Derived
Stromal Cells Inhibits Post-Injury Fibrogenesis Through A JNKDependent Mechanism
Hirotaka Suga,aHitomi Eto,aTomokuni Shigeura,bKeita Inoue,aNoriyuki Aoi,aHarunosuke
Kato,aSatoshi Nishimura,cIchiro Manabe,d,eKoichi Gonda,aand Kotaro Yoshimuraaa

Key words. Adipose-derived stem cells • Hepatocyte growth factor • Fibroblast growth factor-2 • c-Jun N-terminal
kinase • Fibrogenesis • Ischemia-reperfusion injury

ABSTRACT
Background: Adipose-derived stem/stromal cells(ASCs) not only function as tissue-specific progenitor
cells, but are also multipotent and secrete angiogenic
growth factors such as hepatocyte growth factor(HGF) under certain circumstances. However, thebiological role and regulatory mechanism of thissecretion have not been well studied.
Methods and Results: We focused on the role ofASCs in the process of adipose tissue injury andrepair, and found that among injury-associatedgrowth factors, fibroblast growth factor-2 (FGF-2)
strongly promoted ASC proliferation and HGF
secretion through a c-Jun N-terminal kinase (JNK)
signaling pathway. In a mouse model of ischemiareperfusion injury of adipose tissue, regenerativechanges following necrotic and apoptotic changes
were seen for 2 weeks. Acute release of FGF-2 byinjured adipose tissue was followed by upregulationof HGF. During the adipose tissue remodelingprocess, adipose-derived BrdU-positive cells wereshown to be ASCs (CD31–CD34+). Inhibition of JNK
signaling inhibited the activation of ASCs anddelayed the remodeling process. In addition,
inhibition of FGF-2 or JNK signaling prevented post-

injury upregulation of HGF and led to increased
fibrogenesis in the injured adipose tissue. Increased
fibrogenesis also followed the administration of aneutralizing antibody against HGF.
Conclusions: FGF-2 released from injured tissue acts
through a JNK signaling pathway to stimulate ASCs
to proliferate and secrete HGF, contributing to theregeneration of adipose tissue and suppression offibrogenesis after injury. This study revealed afunctional role for ASCs in the response to injury
and provides new insight into the therapeuticpotential of ASCs.

Author contributions: H.S.: Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing; H.E.: Collection and/or
assembly of data; T.S.: Collection and/or assembly of data; K.I.: Collection and/or assembly of data; N.A.: Collection and/or assembly of data; H.K.:
Collection and/or assembly of data; S.N.: Data analysis and interpretation; I.M.: Data analysis and interpretation; K.G.: Data analysis andinterpretation; K.Y.: Conception and design, Financial support, Administrative support, Collection and/or assembly of data, Data analysis andinterpretation, Manuscript writing.
Correspondence: Kotaro Yoshimura, M.D., Department of Plastic Surgery, University of Tokyo School of Medicine, 7-3-1, Hongo, Bunkyo-Ku,
Tokyo 113-8655, Japan. TEL: +81-3-5800-8948, FAX: +81-3-5800-8947, E-mail: yoshimura-pla@h.u-tokyo.ac.jp. This work was supported by agrant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (contact grant number: B2- 19390452). Received March
20, 2008; accepted for publication August 28, 2008; first published online in Stem Cells Express September 4, 2008. ©AlphaMed Press 10665099/2008/$30.00/0 doi: 10.1634/stemcells.2008-0261

Copyright © 2008 AlphaMed Press

Downloaded from www.StemCells.com at Tokyo U Nogaku on September 4, 2008

Department of Plastic Surgery, University of Tokyo, Tokyo, Japan; bDivision of Research and Development,
Biomaster Inc., Kanagawa, Japan; cDepartment of Cardiovascular Medicine, University of Tokyo, Tokyo, Japan;dNano-Bioengineering Education Program, University of Tokyo, Tokyo, Japan; ePRESTO, Japan Science andTechnology Agency, Tokyo, Japan

HGF secretion from ASCs induced by FGF-2

Adipose-derived stem/stromal cells (ASCs)
function as tissue-specific progenitor cells, candifferentiate into cells of various lineages [1],and secrete many potent growth factors andcytokines such as vascular endothelial growth
factor (VEGF) and hepatocyte growth factor(HGF) [2-4]. Paracrine effects of the secreted
factors account for improved vascularity ofischemic hind limbs treated with ASCs [2,4];
however, ASC differentiation into endothelial
cells could contribute to the improved
vascularity as well [5,6]. The use of ASCs forpromoting angiogenesis and tissue repair has
gained interest as a potential therapeuticstrategy [7-9] and clinical trials involving
ASC-mediated enhancement of bone andadipose regeneration and angiogenesis areunderway [10-12].

We hypothesized that ASCs stimulated byfactors released from the injured tissue play animportant role in the repair process of adipose
tissue, perhaps by secreting regenerationassociated growth factors or by differentiating
into adipocytes, endothelial cells, or other cell
types. We examined the influence andmechanisms of injury-related factors on humanand murine ASCs in vitro. Furthermore, using
an original mouse model of ischemiareperfusion injury to adipose tissue, we
assessed the expression of injury-related
growth factors at the cellular and molecularlevel in order to elucidate the role of ASCs
during the regeneration process.

ASCs not only secrete potent growth factors
and cytokines but they are also affected bythem. Fibroblast growth factor-2 (FGF-2)
stimulates the growth of ASCs [13, 14] and has
been shown to promote adipogenic [15] andchondrogenic [13] differentiation of ASCs;
however, FGF-2 inhibits their osteogenic
differentiation [14]. Platelet-derived growth
factor (PDGF) induces proliferation andmigration of ASCs [16]. A culture medium
containing VEGF, FGF-2, epidermal growth
factor (EGF), and insulin-like growth factor-1(IGF-1)markedly
acceleratedASC
proliferation and preserved their multipotency[17], suggesting synergism between thesegrowth factors on ASC growth.

MATERIALS AND METHODS

Mitogen-activated protein kinases (MAPKs)
are important signal transducing enzymes thatare involved in many facets of cellular
regulation by growth factors [18]. Among
MAPKs, c-Jun N-terminal kinases (JNK) play
key roles in PDGF-induced proliferation andmigration of ASCs [16]. There is also a study
demonstrating that ASCs produce VEGF,
HGF, and IGF-1 in response to tumor necrosis
factor-α by a p38 MAPK-dependent
mechanism [19].

Cell isolation and culture
Liposuction aspirates were obtained fromhealthy female donors (mean age, 35.4 ± 3.3;
mean BMI, 22.1 ± 1.2, n = 9) undergoing
liposuction of the abdomen or thighs. Eachpatient provided their informed consent using
an institutional review board-approved
protocol prior to the procedure. ASCs wereisolated from the aspirated fat as described
previously [23]. Briefly, the aspirated fat waswashed with phosphate buffered saline (PBS)
and digested on a shaker at 37ºC in PBS
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In this study, we evaluated the influence of
injury-associated growth factors on ASCs. In
the process of injury and subsequent woundhealing, various growth factors andinflammatory cytokines regulate regenerative
cellular activities. We previously analyzed
wound fluids after liposuction surgery andreported the sequential expression of growth
factors: FGF-2 and PDGF were released in theearly stage of wound healing, while VEGF and
HGF were expressed in the later stage [20].
Administration of growth factors has beenreported to enhance the regeneration of injured
tissues, such as FGF-2 for burn [21] and HGF
for myocardial infarction [22]. However, few
studies have focused on the injury and repair
process in adipose tissue and the potential roleof ASCs, presumably because there is no goodmodel of adipose tissue injury.

INTRODUCTION

HGF secretion from ASCs induced by FGF-2

without 10 ng/ml of various growth factors.
Afterward, BrdU labeling was performed for 3
hours at a final concentration of 10 μM.

Proliferation assay
Human ASCs were plated in a 6-well plate at 2
× 104 cells/well. Each injury-associated growth
factor (VEGF, FGF-2, HGF, and PDGF; allfrom Wako Pure Chemicals, Osaka, Japan) was
added to the control medium (DMEM and 10%
FBS) at concentrations of 0.1, 1, and 10 ng/ml.
Cell number was counted after 3, 6, and 9 daysinculture
using
a
cell
counter(NucleoCounterTM, Chemometec, Allerod,
Denmark). For other types of cells, 2 × 105
cells were plated in a 100-mm dish and cellnumbers were counted after 7 days. 5-bromo2-deoxyuridine (BrdU) incorporation assays
were performed using the BrdU In-Situ
Detection Kit (BD Biosciences, San Diego,
CA). Human ASCs were plated in a 4-wellchamber slide at 5×104 cells/well. Cells were
cultured for 48 hours in control medium with areduced serum concentration (2% FBS) with or

Quantitative real-time reverse-transcriptase
polymerase chain reaction
We isolated RNA from human ASCs cultured
with VEGF, FGF-2, HGF, or PDGF at 10
ng/ml. For other types of cells, RNA was
isolated from cells cultured with or without
FGF-2 (10 ng/ml). We also isolated RNA fromthe inguinal adipose tissue of mouse models(see below) after homogenizing. Two
micrograms of total RNA was isolated using an
RNeasyTM Mini Kit (QIAGEN, Hilden,
Germany), followed by reverse transcription.
We amplified cDNA for 40 cycles with the
ABI 7700 sequence detection system, a
TaqManTM Universal PCR Master Mix, and thefollowing
pre-designed
primersandfluorescein-labeled probes: human VEGF
(Hs00900054_m1); FGF-2 (Hs00266645_m1);
HGF(Hs00300159_m1);
PDGF(Hs00234042_m1);
GAPDH

Flow cytometry
Human ASCs cultured with VEGF, FGF-2,
HGF, or PDGF at 10 ng/ml for 7 days were
examined for surface marker expression using
flow cytometry. The following fluorochromeconjugated monoclonal antibodies were used:anti-CD31-PE, CD34-PE (BD Biosciences),
Flk-1-PE, and Tie-2-PE (R&D Systems,
Minneapolis, MN). Cells were incubated witheach antibody for 30 minutes and thenanalyzed using an LSR II flow cytometry
system (BD Biosciences). Gates were set basedon staining with combinations of relevant andirrelevant antibodies so that no more than 0.1%
of cells were positive using irrelevant
antibodies. Mouse stromal vascular fraction(SVF) cells were separated as described aboveand multi-color flow cytometric analyses were
performed using the following monoclonal
antibodies conjugated to fluorochromes: antiCD31-PE (BD Biosciences), CD34-FITC(eBioscience, San Diego, CA), CD45-PE Cy7(Beckman Coulter, Fullerton, CA), APC BrdU
Flow Kit (BD Biosciences), and Annexin VFITC Apoptosis Detection Kit (BDBiosciences).

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containing 0.075% collagenase for 30 min.
Mature adipocytes and connective tissue wereseparated from pellets by centrifugation (800 ×
g, 10 min). The cell pellets were resuspended,filtered through 100-µm mesh, plated at adensity of 5×105 nucleated cells/100-mm dish,and cultured at 37ºC in an atmosphere of 5%
CO2 in humid air. The culture medium was
Dulbecco’s
modified
Eagle’s
medium(DMEM) containing 10% fetal bovine serum(FBS). Primary cells were cultured for 7 days
and were defined as “Passage 0”. The medium
was replaced every 3 days. Cells werepassaged every week by trypsinization. Human
ASCs at Passage 1 to 3 were used in theexperiments. To separate mouse ASCs, adipose
tissues were obtained from inguinal fat pads of
6-week-old male ICR mice. The adipose
tissues were minced into 2-3-mm pieces andprocessed as described above. Mouse ASCs at
Passage 1 to 3 were used in the experiments.
Human dermal fibroblasts (hDFs) wereobtained from explant cultures of skin samples
from separate donors; hDFs at Passage 3 to 5
were used in the experiments. Human
mesenchymal stem cells from bone marrow(hBM-MSCs), frozen at Passage 2, werepurchased from Cambrex (Walkersville, MD);hBM-MSCs at Passage 3 to 5 were used in theexperiments.

HGF secretion from ASCs induced by FGF-2(Hs99999905_m1);
mouse
VEGF
(Mm00437304_m1);
FGF-2(Mm00433287_m1);
HGF(Mm01135182_m1);
and
GAPDH
(Mm99999915_m1; all primers from Applied
Biosystems, Foster City, CA). We calculated
expression levels by the comparative CT
method using GAPDH as an endogenous
reference gene.
Quantifying HGF protein by enzyme-linked
immunosorbent assay
Conditioned media of human ASCs cultured
with or without FGF-2 (10 ng/ml) for 72 hours
were
analyzedbyenzyme-linked
immunosorbent assay (ELISA) using an
ELISA kit for human HGF (QuantikineTM,
R&D Systems). Data were expressed as thesecreted factor per 106 cells at the time of
harvest.
Inhibition of MAP kinase signaling
pathways
One selective inhibitor for each of threesignaling pathways (ERK inhibitor U0126, p38
proteins inhibitor SB202190, and JNK
inhibitor SP600125; all from Calbiochem, La
Jolla, CA) was added at 10 µM with FGF-2 (10ng/ml), and the effects on proliferation andgene
expression
wereexaminedbyproliferation assay and real-time reverse
transcriptase-polymerase chain reaction (RTPCR).

In vivo inhibition assays
Inhibitor solutions were infused continuously
using an osmotic pump (Model 1007D,ALZET, Cupertino, CA) that released liquid at
a rate of 0.5 µl/h for about 7 days. The pump,containing 100 µl of a JNK inhibitor(SP600125; 10 µM), goat anti-FGF-2 antibody(R&D Systems; 50 µg/ml), goat anti-mouse
HGF antibody (R&D Systems; 50 µg/ml), or
control PBS was implanted subcutaneously at
the time of reperfusion.

JNK activity assay
JNK activity was measured by the extent of cJun phosphorylation using a cell-based ELISA
kit (CASETM Kit, SuperArray, Frederick, MD)
according to manufacturer’s instructions. In
brief, human ASCs were seeded at 2×104 per
well in a 96-well plate and allowed to sit
overnight. Cells were then starved in serumfree medium for 24 hours. Cells werepretreated with vehicle (0.1% DMSO), U0126,
SB202190, or SP600125 for 15 min, then they
were exposed to FGF-2 (10 ng/ml) or HGF (10or 100 ng/ml) for 15 min. The amounts ofactivated (phosphorylated) c-Jun protein andtotal c-Jun protein were measured using antiphospho-c-Jun (Serine 73) antibody and antipan-c-Jun antibody.

Histological detection of apoptosis andproliferation
To detect apoptosis after ischemia-reperfusion
injuryinadiposetissue,terminal
deoxynucleotidyl transferase-mediated dUTP
nick end-labeling (TUNEL) staining was
performed using an In Situ Cell Death
Detection Kit (Roche Diagnostics, Mannheim,
Germany). To detect proliferating cells, 1 mg
of BrdU was administered intraperitoneally 6
and 3 hours before tissue harvest. BrdU4

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Mouse model for ischemia-reperfusion
injury to adipose tissue
Care of animals was in accordance withinstitutional guidelines. Six-week-old ICR
mice were anesthetized with pentobarbital (50mg/kg weight) and a 2-cm incision was madein the inguinal region. The subcutaneous
inguinal fat pad was elevated with the main
nutrient vessels arising from the femoral
vessels intact. Small communicating vessels tothe skin from the fat pad were electrocoagulated. The main vessels were clampedwith a vessel micro-clip for three hours andthen released to allow reperfusion. The adipose
tissue samples were harvested at various
intervals (at days 1, 3, 7, and 14) after
ischemia-reperfusion injury, and examined byflow cytometry, real-time RT-PCR, western
blotting, and histology (hematoxylin-eosin
staining, Azan staining and others; see below).
Fat samples from sham-operated animals(without ischemia-reperfusion injury) wereused as controls. At each time point we
confirmed that contralateral pads fromexperimental animals showed no pathologicalchanges as compared to the sham-operated
animal samples (“day 0” sample).

HGF secretion from ASCs induced by FGF-2

GAPDH signal served as an internal control.
Protein bands were quantified by volume
summation of image pixels using Photoshop
7.0 (Adobe Systems Inc., San Jose, CA).

positive cells were detected using a BrdU InSitu Detection Kit (BD Biosciences). The
number of TUNEL or BrdU positive cells wascounted at a 200× magnification using threerandomly selected fields per section.

Living tissue imaging
Visualization of living adipose tissue was
performed using the procedure of Nishimura etal [25]. Briefly, the adipose tissue was minced
into 3-mm pieces and incubated with thefollowing reagents for 30 min: BODIPYTM
558/568 or BODIPYTM-FL (both from
Molecular Probes) to stain adipocytes, Alexa
FluorTM 488-conjugated isolectin GS-IB4(Molecular Probes) to stain endothelial cells,
Hoechst 33342 (Dojindo, Kumamoto, Japan) tostain all nulei, or propidium iodide (PI; SigmaAldrich, St. Louis, MO) to stain nuclei of
necrotic cells. The sample was then washedand directly observed with a confocal
microscope system (Leica TCS SP2).

Western blotting
Adipose tissue specimens were homogenizedin 1ml of lysis buffer (Santa Cruz
Biotechnology) and centrifuged at 15,000 rpm
for 2 minutes. The aqueous layer was collected
and the protein concentration was determinedusing the BCA protein assay kit (Pierce,
Rockford, IL). Equal amounts of protein (10μg) were loaded into each lane of an SDSPAGE gel. The resolved proteins weretransferred to a PVDF membrane (Bio-Rad
Laboratories,
Hercules,CA)andimmunostaining was performed using goatanti-mouse FGF-2 antibody (R&D Systems),
goat anti-mouse HGF antibody (R&D
Systems), and goat anti-mouse GAPDH
antibody (Santa Cruz Biotechnology). The

Statistical analysis
Results were expressed as mean ± standard
error of means (SEM). Comparisons between
two groups were performed with Welch’s ttest. Comparisons of multiple groups weredone by analysis of variance with corrections
for multiple comparisons. A value of p < 0.05
was considered significant.

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Glycerol-3-phosphate dehydrogenase(GPDH) assay
A GPDH Assay Kit (Cell Garage, Tokyo,
Japan) was used according to the
manufacturer’s instructions, as previously
described [24]. In brief, each adipose tissue
sample was mixed with a 0.25 M sucrose
solution to a total of 5 ml, homogenized, andcentrifuged. The supernatants were diluted 10
times with an enzyme-extracting reagent andthe optical absorption was measured at 340 nm
for 10 minutes in a 96-well plate after additionof twice the volume of a substrate reagent.
GPDH activity was calculated based on thefollowing formula: GPDH activity (U/ml) =ΔOD × 0.482 × 10 (ΔOD: change in opticaldensity per minute).

Immunohistochemistry
Harvested adipose tissue samples were zincfixed (Zinc Fixative, BD Biosciences) andparaffin-embedded. We prepared 6-µm-thicksections and performed immunostaining using
the following primary antibodies: goat antiFGF-2 (R&D Systems), goat anti-mouse HGF(R&D Systems), goat anti-mouse CD68 (Santa
Cruz Biotechnology, Santa Cruz, CA), goatanti-mouse CD34 (Santa Cruz Biotechnology),
rat anti-mouse CD31 (BD Biosciences), andbiotinylatedmouse
anti-BrdU(BD
Biosciences).
Forvisualization
withdiaminobenzidine(DAB),
peroxidaseconjugated secondary antibodies appropriate
for each primary antibody (Nichirei
Biosciences, Tokyo, Japan) or a streptavidinperoxidase complex (BD Biosciences) wereused. For a double fluorescence staining, thefollowing secondary antibodies or reagent wereused: Alexa FluorTM 488-conjugated rabbit
anti-rat IgG, Alexa FluorTM 488 or 568conjugated donkey anti-goat IgG, and Alexa
FluorTM
568-conjugatedstreptavidin(Molecular Probes, Eugene, OR). Isotypic
antibody was used to serve as a negative
control for each staining.

HGF secretion from ASCs induced by FGF-2

Effects of injury-associated growth factors
on human ASC proliferation
FGF-2 and PDGF promoted proliferation ofhuman ASCs in a dose-dependent manner,
while VEGF and HGF did not promote ASC
growth (Fig. 1A). Morphologically, ASCs
cultured with FGF-2 were smaller than ASCs
cultured with other growth factors (Fig. 1B).
The proliferative effects of FGF-2 and PDGF
were also demonstrated in a BrdU
incorporation assay. Cells were cultured in
growth factor-containing medium with areduced serum concentration (2% FBS) for a
fixed period (48 hours) prior to assay(Supplemental online Fig. 1A).
Phenotypic effects of injury-associatedgrowth factors on human ASCs
Flow cytometry showed no significant changes
in expressions of endothelial surface markers(CD31, CD34, Flk-1, and Tie-2) by any injuryassociated growth factors, indicating that
supplementation of a single factor is notenough to induce endothelial differentiation ofhuman ASCs, at least when cultured without
any extracellular matrices (Fig. 1C andSupplemental online Fig. 1B).

FGF-2-induced effects in human ASCs andother cell types
Cell growth was promoted by FGF-2 in all fourcell types studied (human ASCs, human DFs,
human BM-MSCs, and murine ASCs),
although the basal proliferative capacity
differed among cell types. The FGF-2enhanced cell proliferation was significantly
inhibited by a JNK inhibitor (SP600125) in all
cell types examined (Fig. 3D). HGF mRNA
expression was also promoted by FGF-2 in all
cell types, and the upregulation of HGF mRNAwas significantly inhibited by the JNK
inhibitor except in human BM-MSCs (Fig. 3E),
suggesting that HGF mRNA upregulation by
FGF-2 was mediated through signaling
pathways other than JNK in human BM-MSCs.

Interactive effects of injury-associatedgrowth factors on their expressions byhuman ASCs
FGF-2 and PDGF significantly downregulated
expression of their own transcripts by humanASCs, and FGF-2 also significantly suppressed
PDGF mRNA expression (Fig. 2A). Most
interestingly, FGF-2 promoted a striking
increase in HGF mRNA expression. Timecourseevaluation
revealedthat
theupregulation of HGF mRNA was biphasic,
with the first peak at 6 hours and a striking
increase beginning after 24 hours (Fig. 2B).
ELISA of culture media demonstrated that
HGF protein secretion by FGF-2-stimulated
human ASCs was significantly elevated at day
3 (Fig. 2C).

Ischemia-reperfusion injury and subsequent
repair in the inguinal adipose tissue of mice
The murine inguinal fat pad consistently had adominant feeding artery and vein without
anatomical
anomaly,whichenabledreproducible
ischemia-reperfusion
injury6

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Intracellular signaling pathways of FGF-2induced effects on human ASCs
The proliferative effects of FGF-2 on humanASCs were significantly inhibited by a JNK
inhibitor (SP600125) but not by an ERK
inhibitor (U0126) or a p38 inhibitor(SB202190; Fig. 3A). Upregulation of HGF
mRNA by FGF-2 was also significantly
inhibited by the JNK inhibitor (Fig. 3B).
Administration of the ERK and p38 inhibitorsslightly decreased the expression level of HGF
mRNA although the changes were notstatistically significant, suggesting crosstalk
between JNK and other signaling pathways.
Phosphorylation of c-Jun, the prototypical
nuclear effector of the JNK signal transduction
pathway, increased with FGF-2 treatment, andthe FGF-2-induced upregulation of c-Jun
phosphorylation was completely prevented by
pretreatment with the JNK inhibitor (Fig. 3C).
On the other hand, HGF did not increase
phosphorylation of c-Jun in human ASCs (Fig.
3C). These results indicate that FGF-2
promotes proliferation and HGF mRNA
expression predominantly through the JNK
signaling pathway.

RESULTSHGF secretion from ASCs induced by FGF-2

Cellular events in the repair process of
injured adipose tissue
Before injury, CD34+/CD31– cells, whichwere regarded as ASCs, were found throughout
the intact adipose tissue, located between
mature adipocytes, and especially abundantaround vessels (Supplemental online Fig 3A).
The number of CD34+/CD31– cells wasapparently much larger than that of CD31+
endothelial cells, which were negative or
faintly positive for CD34. Most BrdU-positive
proliferating cells, frequently detected 3 days
after injury, were also positive for CD34 (Fig.
6A, top) and most CD34+ cells were lectinnegative (Fig. 6A, bottom), which suggests that
CD34+/lectin– ASCs play substantial roles in
the repair process. Living tissue imaging
revealed increase of interstitial space andsmall-sized adipocytes (less than 50 µm in
diameter), an increased number of nucleatedcells including lectin-positive round cells (Fig.
6B), and capillaries especially around smallsized adipocytes (Supplemental online Fig. 3B).
On day 7, large-sized adipocytes were not seen

FGF-2 protein was detected in the interstitial
tissue by immunohistochemistry as early as
day 1, with a peak of detection on day 3,
although not much FGF-2 was expressed on
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day 7 (Fig. 5A). Western blotting also showed
an increase in FGF-2 protein on days 1 and 3(Fig. 5B). On the other hand, little HGF protein
was detected on day 1, but HGF expression
was elevated from day 3 until at least day 7(Fig. 5C and 5D). Interestingly, FGF-2 mRNAwas downregulated from soon after injury
throughout the 2 weeks, suggesting that FGF-2
was released from the storage of the injured
adipose tissue, such as extracellular matrix ordying cells, rather than produced by viable
cells such as ASCs. The released FGF-2 might
downregulate FGF-2 mRNA expression byASCs in the injured adipose tissue, as was seen
in human ASCs in vitro. In contrast, HGF
mRNA expression in the tissue wassignificantly elevated on day 3, but not on days
1 and 7 (Fig. 5E), suggesting that the delayed
upregulation of HGF mRNA and secretion of
HGF were likely induced by the preceding
release of FGF-2 protein. Thus, the datasuggest that FGF-2 is provided temporarily
only when an event such as injury occurs andtriggers FGF-JNK-HGF signaling, thereby
promotingrepairprocesses
suchasangiogenesis.

experiments (Fig. 4A). The weight of theadipose tissue was increased on day 1,
suggesting tissue edema (Supplemental online
Fig 2A), while GPDH activity, whichcorrelates with the total number and size ofmature adipocytes, did not change significantly
during the experimental period (Supplemental
online Fig. 2B). Histologically, interstitial
infiltration of blood cells was observed as early
as day 1 and most prominent on day 3. Smallsized adipocytes appeared on day 1 andincreased in number on day 3, suggesting thatlipolysis or adipogenesis took place throughout
the adipose tissue. On day 7, infiltrated
erythrocytes disappeared and some adipocytes
increased in size while a substantial number ofnucleated cells remained in the interstitialspace between adipocytes (Fig. 4B). Flowcytometric analysis of the SVF showed that the
total number of SVF cells as well as thenumber of CD45+ cells was increased by day
7, while the number of CD34+/CD31– cells
peaked on day 3 (Fig. 4C). The number of
BrdU-positive proliferating cells increased
from day 1 and peaked on day 3 (Fig. 4D and
Supplemental online Fig. 2C), and most
BrdU+/CD45– cells (88.2 ± 4.2%, n = 3) wereshown to be CD34+/CD31– (Fig. 4E). The
frequency of TUNEL-positive apoptotic cells
was significantly high (6-9% of nucleatedcells) on day 1 and decreased thereafter (Fig.
4F and Supplemental online Fig. 2D). Flowcytometry demonstrated that most Annexin Vpositive apoptotic cells on day 1 (90.1 ± 3.0%,
n = 3) were CD45+ (Fig. 4G). In addition, an
increase in PI-positive necrotizing cells wasobserved on day 3; some of these cells (17.2 ±
5.5%, n = 3) were lectin-positive, suggestingthat some capillary endothelial cells were
necrotizing and that capillary remodeling wasunderway (Fig. 4H). These results indicate thatthe adipose tissue was impaired soon after
reperfusion, apoptosis and necrosis wereinvolved in the process of adipose
degeneration, and regenerative changes
occurred thereafter.

HGF secretion from ASCs induced by FGF-2

administration of vehicle alone. Significantly
increased fibrogenesis was also observed in thegroup treated with an anti-HGF antibody (Fig.
7E). These results indicated that fibrogenesis
seen in the injured adipose tissue wassuppressed by HGF in the process of adipose
tissue repair and suggested that HGF secretion,
mainly from ASCs, was induced by FGF-2
through a JNK signaling pathway.

in samples treated with a JNK inhibitor, whilelarge-sized adipocytes were seen in untreatedmodels (Fig. 6B and 6C). Furthermore, PIpositive nuclei were frequently observed ondays 1 and 3, a small number of which (10.7 ±
5.7% on day 1; 4.4 ± 0.5% on day 3; n = 3)
were seen in adipocytes, suggesting that not
only capillary endothelial cells (Fig. 4H) but
also adipocytes were necrotizing (Fig. 6D). On
day 7, the PI-positive nuclei were still observedin samples treated with a JNK inhibitor,
although they were rarely detected in untreatedmodels (Fig. 6D and 6E). These results suggestthat the adipose tissue remodeling involving
adipocytes and capillaries was underway
during the first week after injury and the tissue
repair process was impaired by treatment witha JNK inhibitor. CD68+ cells, regarded as
infiltrating macrophages, were scarcely
detected in the intact adipose tissue(Supplemental online Fig. 3C). However,infiltrated macrophages aggregated aroundsmall-sized adipocyte-like cells after injury,
suggesting phagocytosis. This aggregation of
CD68+ cells was most frequently observed 3days after injury.

DISCUSSION

Influence of signal inhibition on HGF
expression and fibrogenesis after ischemiareperfusion injury
The increase in BrdU-positive proliferating
cells on day 3 was significantly inhibited bytreatment with a JNK inhibitor or aneutralizing antibody against FGF-2, but notby a neutralizing antibody against HGF (Fig.
7A).
Immunohistochemical
analysisdemonstrated that the JNK inhibitor also
suppressed the number of CD34+/lectin– ASCson days 3 and 7 (Supplemental online Fig. 3D).
Administering a JNK inhibitor or a neutralizing
antibody against FGF-2 completely prevented
upregulation of HGF mRNA in the injured
adipose tissue on day 3 (Fig. 7B) andsuppressed HGF secretion on day 7 (Fig. 7C
and 7D). Histological measurement of the
fibrous area at 2 weeks revealed thatreperfusion injury to the adipose tissue caused
a significant increase in the fibrous area (from
10% to 23%). Furthermore, inhibition of JNKor FGF-2 signaling caused a further significant
increase in the fibrous area as compared to8

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After mechanical injury to human adipose
tissue, FGF-2, EGF, transforming growth
factor (TGF)-β, and PDGF are first secreted in
the early stage of wound healing. Thereafter, as
the above growth factors decline, VEGF and
HGF secretion gradually increases during thefirst week post-injury [20]. Our results
indicated that, among these injury-associatedgrowth factors, FGF-2 promoted proliferationand HGF secretion by ASCs predominantly
through a JNK signaling pathway. FGF-2 is an
importantendogenous
stimulatorofangiogenesis [26] and cell proliferation [27]and is known to be released during the earlyphase of wound healing [28,29]. Cellular FGF2 is released during the lysis of various cell
types, such as fibroblasts [30] and endothelial
cells [31], around the wound, while FGF-2
bound up in the extracellular matrix is releasedby the action of various wound proteases[32,33]. JNK signaling is involved not only in
FGF-2-induced ASC proliferation, as revealedin this study, but also in PDGF-induced
proliferation and migration of human ASCs
[16]. Therefore, it is likely that the JNK
inhibitor used in this study inhibited thebiological action of both FGF-2 and PDGF on
ASCs. PDGF promoted ASC proliferation, but
had no significant effect on HGF expression by
ASCs. As other researchers have reported, both
FGF-2 and EGF promoted HGF secretion byASCs [3]; therefore, JNK might also be
involved in the EGF-induced HGF secretion by
ASCs. Although some studies using other cell
types found that JNK was involved in the HGF
signaling pathway [34,35], our results
indicated that HGF, even at highconcentrations, did not stimulate the JNK
pathway in human ASCs.

HGF secretion from ASCs induced by FGF-2

The injury and repair process in adipose tissue
has not been well studied, presumably because
there is no standard animal model. Coban etal., using the epigastric adipo-cutaneous flap
model, described edema and hemorrhage
following ischemia-reperfusion injury of
adipose tissue [43]; their results were similar toours, even though a different model was used.
In the injured adipose-tissue, necrotic andapoptotic changes followed edema andhemorrhage in the early phase after injury, andinflammatory and regenerative changes such as
phagocytosis, cell infiltration and proliferation
were observed throughout the first week. The
necrotic changes were seen in capillary
endothelial cells and adipocytes, suggesting
adipose tissue remodeling, the repair process ofwhich was impaired by a JNK inhibitor. HGF
secretion followed release of FGF-2, stored
bound or in other forms, from the tissue; the
HGF upregulation appears to be derived
predominantly from ASCs stimulated by FGF2 via JNK signaling pathway, because ASCs
are known to constitute more than one-half ofmesenchymal cells in stromal vascular
fractions derived from adipose tissue [23]. The
ischemia-reperfusion model used in this study
provided reproducible results of injury andregeneration of adipose tissue, all of whichwere complete within 2 weeks.

HGF has been reported to have antifibrogenic
effects in various organs including heart [22],liver [45], and kidney [46]. Recent studies
9

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In the adipose tissue, CD34+/CD31– cells,
which were suggested to be ASCs, were seenin abundance between adipocytes (closely
related to capillaries) and around vessel walls(especially in the tunica adventitia) before
injury. CD34+ vascular wall resident
progenitor cells, which have the capacity todifferentiate into vascular endothelial cells andform capillary sprouts, were previously
reported [43] and this population might
constitute part of ASCs. On the other hand, aperiendothelial subpopulation of ASCs with
pericytic characteristics has been alsocharacterized [44]. ASCs might consist ofheterogenic subpopulations, which may
explain the complexity in elucidating ASC
characteristics and functions in vivo. As
previously suggested, there is a close
relationship between adipogenesis andangiogenesis, therefore, ASCs, which areprimarily regarded as adipocyte progenitor
cells, may function as adipogenic andangiogenic progenitors [5] and manage theinterplay between adipocytes, blood cells, andblood vessels in various situations, such as
obesity [25], adipose tissue turnover, and postinjury adipose tissue repair. During tissue
regeneration, more CD34+/CD31– ASCs were
found between adipocytes than in controls, andmost BrdU-positive proliferating cells seen on
day 3 were CD34+/lectin–, suggesting that
ASCs are proliferating and participating inrepair processes such as adipogenesis andangiogenesis. The proliferation and migrationof ASCs were strongly inhibited by treatmentof a JNK inhibitor, resulting in delayed andimpaired tissue repair as well as increased
fibrogenesis. Macrophages are known to be
involved in inflammation in adipose tissue andobesity [25]. In this study, CD68+
macrophages were scarcely detected incontrols but were seen in larger numbers on
day 3, however, at a much lower level than inASCs. The role of macrophages in the injured
adipose tissue remains to be clarified, but they
may be involved in phagocytosis and/or
angiogenesis [25].

It is interesting that the FGF-2-enhanced cellproliferation and HGF production that we
observed for ASCs were observed also in othermesenchymal cell types, such as BM-MSCs
and DFs, although HGF mRNA upregulationin human BM-MSCs was mediated by signals
other than JNK. FGF-2-induced HGF
upregulation has been observed in other cell
types, such as smooth muscle cells [36],fibroblast-like cells from lung tissue [37], andosteoblasts [38]; however, the intracellularsignaling mechanisms were not wellinvestigated. Other factors such as interleukin1 [39], interferon-γ [40], and ascorbic acid [41]have also been reported to stimulate HGF
production. HGF is a key mediator ofangiogenesis and wound healing, and its
expression seems to be regulated by a numberof factors in various cells and tissues.

HGF secretion from ASCs induced by FGF-2

CONCLUSIONS
FGF-2 promotes proliferation of and HGF
secretion by ASCs through a JNK signaling
pathway. In injured adipose tissue, ASCs
stimulated by FGF-2 released from injured
tissue are suggested to be the main
proliferating cell population in the adipose
repair process. This JNK-mediated signal plays
an important role in preventing fibrogenesis
and preserving adipose volume after injury.
This study also revealed a new role for ASCs
in the injury response and provides insights
into future strategies for ASC-based therapies.

The results of this study provide new insights
into the therapeutic potential of ASCs.
Cytotherapies with ASCs, especially whenpretreated or combined with FGF-2, might
accelerate tissue repair, reduce fibrosis, andimprove the function of organs impaired byacute or chronic inflammation through HGF
action. It has been suggested that HGF secretedby ASCs might play an important role inautologous fat grafting [9,12]; promoting
adipose graft survival and preventing scarformation. HGF is not only antifibrogenic but
it is also angiogenic [49], and has been shownto contribute to angiogenesis in adipose tissue[50] and to the improvement of ischemic limb[51]. FGF-2-induced HGF secretion could also

ACKNOWLEDGMENTS
We thank Ayako Kurata and Akiko Matsuura
for their technical assistance. This work was
supported by a grant from the Japanese
Ministry of Education, Culture, Sports,
Science, and Technology (contact grant
number: B2- 19390452).

adipose stromal cells. Circulation 2004; 109: 12921298.
3. Kilroy GE, Foster SJ, Wu X, et al. Cytokine profile ofhuman adipose-derived stem cells: Expression ofangiogenic, hematopoietic, and pro-inflammatory
factors. J Cell Physiol 2007; 212: 702-709.
4. Nakagami H, Maeda K, Morishita R, et al. Novel
autologous cell therapy in ischemic limb disease
through growth factor secretion by cultured adipose

REFERENCES
1. Zuk PA, Zhu M, Ashjian P, et al. Human adipose
tissue is a source of multipotent stem cells. Mol Biol
Cell 2002; 13: 4279-4295.
2. Rehman J, Traktuev D, Li J, et al. Secretion ofangiogenic and antiapoptotic factors by human10

Downloaded from www.StemCells.com at Tokyo U Nogaku on September 4, 2008

promote angiogenesis during tissue repair. The
total capillary amount was increased but thevascular density in the injured adipose tissue
was not (Supplemental online Fig. 3E), though
the measurement of vascular density may have
limitation because it can be affected by the sizeand density of adipocytes. In ASCs, VEGF
secretion appears to be induced by hypoxia [2].
The model we used in this study was an injury
model rather than an ischemia model, andVEGF expression was downregulated soonafter injury (data not shown). To study
angiogenesis in adipose tissue and establish atherapeuticstrategyforenhancingangiogenesis, studies using specific models ofchronic ischemia in adipose tissue are needed..

revealed that HGF antagonizes the profibrotic
actions of TGF-β by intercepting Smad signaltransduction through diverse mechanisms[46,47]. In this study, inhibiting FGF-2
signaling suppressed HGF production byASCs, and inhibition of HGF led to increased
fibrogenesis in the injured adipose tissue.
Furthermore, administering a JNK inhibitor oran anti-FGF-2 antibody also resulted in severe
post-injury fibrogenesis, which means that
FGF-2-induced prevention of fibrogenesis wasmediated at least in part by HGF derived from
ASCs and that HGF is a key factor inpreventing fibrogenesis and preserving adipose
volume after injury. FGF-2 is known topromote wound healing by its direct effects on
fibroblasts and endothelial cells [48], but thisstudy revealed that FGF-2 could also promote
tissue repair and prevent fibrogenesis by
inducing HGF from ASCs and/or DFs.
Because adipose tissue exists in or around mostorgans, FGF-2-induced HGF secretion from
ASCs might be important in the repair
processes following organ injuries such as
myocardial infarction, hepatitis, and nephritis.

HGF secretion from ASCs induced by FGF-2

by a p38 MAPK-dependent mechanism. Am J
Physiol Regul Integr Comp Physiol 2006; 291:
R880-R884.
20. Aiba-Kojima E, Tsuno NH, Inoue K, et al.
Characterization of wound drainage fluids as asource of soluble factors associated with woundhealing: Comparison with platelet-rich plasma andpotential use in cell culture. Wound Repair Regen
2007; 15: 511-520.
21. Akita S, Akino K, Imaizumi T, et al. A basic
fibroblast growth factor improved the quality of skingrafting in burn patients. Burns 2005; 31: 855-858.
22. Nakamura T, Mizuno S, Matsumoto K, et al.
Myocardial protection from ischemia/reperfusion
injury by endogeneous and exogenous HGF. J Clin
Invest 2000; 106: 1511-1519.
23. Yoshimura K, Shigeura T, Matsumoto D, et al.
Characterization of freshly isolated and cultured cells
derived from the fatty and fluid portions ofliposuction aspirates. J Cell Physiol 2006; 208: 6476.
24. Suga H, Matsumoto D, Inoue K, et al. Numericalmeasurement of viable and nonviable adipocytes andother cellular components in aspirated fat tissue.
Plast Reconstr Surg 2008; 122: 103-114.
25. Nishimura S, Manabe I, Nagasaki M, et al.
Adipogenesis in obesity requires close interplay
between differentiating adipocytes, stromal cells, andblood vessels. Diabetes 2007; 56: 1517-1526.
26. Montesano R, Vassalli JD, Baird A, et al. Basic
fibroblast growth factor induces angiogenesis invitro. Proc Natl Acad Sci USA 1986; 83: 7297-7301.
27. Nissen NN, Polverini PJ, Gamelli RL, et al. Basic
fibroblast growth factor mediates angiogenic activity
in early surgical wounds. Surgery 1996; 119: 457465.
28. Cordon-Cardo C, Vlodavsky I, Haimovitz-Friedman
A, et al. Expression of basic fibroblast growth factor
in normal human tissues. Lab Invest 1990; 63: 832840.
29. Schulze-Osthoff K, Risau W, Vollmer E, et al. In
situ detection of basic fibroblast growth factor byhighly specific antibodies. Am J Pathol 1990; 137:
85-92.
30.
Takimiya
M,
SaigusaK,
Aoki
Y.
Immunohistochemical study of basic fibroblast
growth factor and vascular endothelial growth factor
expression for age determination of cutaneous
wound. Am J Forensic Med Pathol 2002; 23: 264267.
31. Muthukrishnan L, Warder E, McNeil PL. Basic
fibroblast growth factor is efficiently released from acytolsolic storage site through plasma membrane
disruptions of endothelial cells. J Cell Physiol 1991;
148: 1-16.
32. Bashkin P, Doctrow S, Klagsburn M, et al. Basic
fibroblast growth factor binds to subendothelial
extracellular matrix and is released by heparitinaseand heparin-like molecules. Biochemistry 1989; 28:
1737-1743.
33. Ishai-Michaeli R, Eldor A, Vlodavsky I. Heparanase
activity expressed by platelets, neutrophils, and11

Downloaded from www.StemCells.com at Tokyo U Nogaku on September 4, 2008

tissue-derived stromal cells. Arterioscler Thromb
Vasc Biol 2005; 25: 2542-2547.
5. Planat-Benard V, Silvestre JS, Cousin B, et al.
Plasticity of human adipose lineage cells toward
endothelial cells: Physiological and therapeutic
perspectives. Circulation 2004; 109: 656-663.
6. Miranville A, Heeschen C, Sengenes C, et al.
Improvement of postnatal neovascularization byhuman adipose tissue-derived stem cells. Circulation
2004; 110: 349-355.
7. Miyahara Y, Nagaya N, Kataoka M, et al.
Monolayered mesenchymal stem cells repair scarred
myocardium after myocardial infarction. Nat Med
2006; 12: 459-465.
8. Rodriguez AM, Pisani D, Dechesne CA, et al.
Transplantation of a multipotent cell population fromhuman adipose tissue induces dystrophin expression
in the immunocompetent mdx mouse. J Exp Med
2005; 201: 1397-1405.
9. Matsumoto D, Sato K, Gonda K, et al. Cell-assisted
lipotransfer: supportive use of human adiposederived cells for soft tissue augmentation withlipoinjection. Tissue Eng 2006; 12: 3375-3382.
10. Lendeckel S, Jodicke A, Christophis P, et al.
Autologous stem cells (adipose) and fibrin glue used
to treat widespread traumatic calvarial defects: casereport. J Craniomaxillofac Surg 2004; 32: 370-373.
11. Garcia-Olmo D, Garcia-Arranz M, Herreros D, et al.
A phase I clinical trial of the treatment of Crohn’sfistula by adipose mesenchymal stem celltransplantation. Dis Colon Rectum 2005; 48: 14161423.
12. Yoshimura K, Sato K, Aoi N, et al. Cell-assisted
lipotransfer (CAL) for cosmetic breast augmentation–supportive use of adipose-derived stem/stromalcells–. Aesthetic Plast Surg 2008; 32: 48-55.
13. Chiou M, Xu Y, Longaker MT. Mitogenic andchondrogenic effects of fibroblast growth factor-2 inadipose-derived mesenchymal cells. Biochem
Biophys Res Commun 2006; 343: 644-652.
14. Quarto N, Longaker MT. FGF-2 inhibits
osteogenesis in mouse adipose tissue-derived stromalcells and sustains their proliferative and osteogenic
potential state. Tissue Eng 2006; 12: 1-14.
15. Kakudo N, Shimotsuma A, Kusumoto K. Fibroblast
growth factor-2 stimulates adipogenic differentiationof human adipose-derived stem cells. Biochem
Biophys Res Commun 2007; 359: 239-244.
16. Kang YJ, Jeon ES, Song HY, et al. Role of c-Jun Nterminal kinase in the PDGF-induced proliferationand migration of human adipose tissue-derived
mesenchymal stem cells. J Cell Biochem 2005; 95:1135-1145.
17. Suga H, Shigeura T, Matsumoto D, et al. Rapid
expansion of human adipose-derived stromal cells
preserving multipotency. Cytotherapy 2007; 9: 738745.
18. Chang L, Karin M. Mammalian MAP kinasesignaling cascades. Nature 2001; 410: 37-40.
19. Wang M, Crisostomo PR, Herring C, et al. Humanprogenitor cells from bone marrow or adipose tissue
produce VEGF, HGF, and IGF-1 in response to TNF

HGF secretion from ASCs induced by FGF-2

42. Coban YK, Kurutas EB, Ciralik H. Ischemiareperfusion injury of adipofascial tissue: An
experimental study evaluating early histologic andbiochemical alterations in rats. Mediators Inflamm
2005; 5: 304-308.
43. Zengin E, Chalajour F, Gehling UM, et al. Vascularwall resident progenitor cells: a source for postnatal
vasculogenesis. Development 2006; 133:1543-1551.
44. Traktuev D, Merfeld-Clauss S, Li J, et al. A
population of multipotent CD34-positive adipose
stromal cells share pericyte and mesenchymal
surface markers, reside in a periendothelial location,and stabilize endothelial networks. Circ Res 2008;
102: 77-85.
45. Ueki T, Kaneda Y, Tsutsui H, et al. Hepatocyte
growth factor gene therapy of liver cirrhosis in rats.
Nat Med 1999; 5: 226-230.
46. Liu Y. Hepatocyte growth factor in kidney fibrosis:
Therapeutic potential and mechanisms of action. Am
J Physiol Renal Physiol 2004; 287: 7-16.
47. Yang J, Dai C, Liu Y. Hepatocyte growth factor
suppresses renal interstitial myofibroblast activationand intercepts Smad signal transduction. Am J Pathol
2003; 163: 621-632.
48. Bikfalvi A, Klein S, Pintucci G, et al. Biologicalroles of fibroblast growth factor-2. Endocr Rev 1997;
18: 26-45.
49. Conway K, Price P, Harding KG, et al. The
molecular and clinical impact of hepatocyte growth
factor, its receptor, activators, and inhibitors inwound healing. Wound Repair Regen 2006; 14: 210.
50. Bell LN, Cai L, Johnstone BH, et al. A central rolefor hepatocyte growth factor in adipose tissue
angiogenesis. Am J Physiol Endocrinol Metab 2008;
294: E336-E344.
51. Cai L, Johnstone BH, Cook TG, et al. Suppression ofhepatocyte growth factor production impairs theability of adipose-derived stem cells to promoteischemic tissue revascularization. Stem Cells. 2007;
25: 3234-3243.

12

Downloaded from www.StemCells.com at Tokyo U Nogaku on September 4, 2008

lymphoma cells releases active fibroblast growth
factor from extracellular matrix. Cell Regul 1990; 1:
833-842.
34. Mori S, Matsuzaki K, Yoshida K, et al. TGF-β and
HGF transmit the signals through JNK-dependent
Smad2/3 phosphorylation at the linker regions.
Oncogene 2004; 23: 7416-7429.
35. Rush S, Khan G, Bamisaiye A, et al. C-jun aminoterminal kinase and mitogen activated protein kinase
1/2 mediate hepatocyte growth factor-induced
migration of brain endothelial cells. Exp Cell Res
2007; 313: 121-132.
36. Onimaru M, Yonemitsu Y, Tanii M, et al. Fibroblast
growth factor-2 gene transfer can stimulate
hepatocyte growth factor expression irrespective ofhypoxia-mediated downregulation in ischemic limbs.
Circ Res 2002; 91: 923-930.
37. Roletto F, Galvani AP, Cristiani C, et al. Basic
fibroblast growth factor stimulates hepatocyte
growth factor/scatter factor secretion by humanmesenchymal cells. J Cell Physiol 1996; 166: 105111.
38. Blanquaert F, Delany AM, Canalis E. Fibroblast
growth factor-2 induces hepatocytes growth
factor/scatter factor expression in osteoblasts.
Endocrinology 1999; 140: 1069-1074.
39. Matsumoto K, Okazaki H, Nakamura T. Upregulation of hepatocyte growth factor gene
expression by interleukin-1 in human skinfibroblasts. Biochem Biophys Res Commun 1992;
188: 235-243.
40. Takami Y, Motoki T, Yamamoto I, et al. Synergistic
induction of hepatocyte growth factor in human skinfibroblasts by the inflammatory cytokinesinterleukin-1 and interferon-γ. Biochem Biophys Res
Commun 2005; 327: 212-217.
41. Wu YL, Gohda E, Iwao M, et al. Stimulation ofhepatocyte growth factor production by ascorbic acidand its stable 2-glucoside. Growth Horm IGF Res
1998; 8: 421-428.

HGF secretion from ASCs induced by FGF-2

Figure 1. Effects of injury-associated growth factors on proliferation and surface marker
expression of human ASCs.
(A) ASC counts after 3, 6, or 9 days culture with VEGF, FGF-2, HGF, or PDGF. FGF-2 and PDGF
promoted proliferation of human ASCs in a dose-dependent manner, while VEGF and HGF
showed no proliferative effects (n = 4; *p < 0.05). (B) Light microscopy photographs of humanASCs cultured with each growth factor for 6 days. Cells cultured with FGF-2 (10 ng/ml) were
smaller than those cultured with other growth factors. Scale bars = 200 µm. (C) Flow cytometricanalyses of human ASCs cultured with each growth factor for 7 days. No significant changes in theexpression of vascular endothelial markers (CD31, CD34, Flk-1, and Tie-2) were observed.

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HGF secretion from ASCs induced by FGF-2

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HGF secretion from ASCs induced by FGF-2

Figure 2. Interactive effects of injury-associated growth factors on their own mRNA expression byhuman ASCs.
(A) Injury-associated growth factor mRNA expression by human ASCs. FGF-2 (10 ng/ml) induced
striking upregulation of HGF mRNA expression by human ASCs, while FGF-2 and PDGF
downregulated their own expression (n = 7, *p < 0.05). (B) Time course of HGF mRNA expression
induced by FGF-2. FGF-2 (10 ng/ml) promoted biphasic HGF mRNA expression by human ASCs,
with the first peak occurring at 6 h and the second increase beginning after 24 h (n = 6, *p < 0.05).
(C) FGF-2-induced HGF protein secretion by human ASCs. ELISA of cultured media on day 7
showed that FGF-2 (10 ng/ml) promoted secretion of HGF protein from human ASCs (n = 4, *p <0.05).

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HGF secretion from ASCs induced by FGF-2

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HGF secretion from ASCs induced by FGF-2

Figure 3. Inhibition of FGF-2 intracellular signaling pathways.

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(A) Effects of downstream signal inhibitors on ASC counts after 7 days in culture with or without
FGF-2 (10 ng/ml). The JNK inhibitor SP600125 (SP) significantly inhibited the proliferative effectof FGF-2 on human ASCs, while U1026 (an ERK inhibitor) and SB202190 (SB, a p38 protein
inhibitor) showed no or limited inhibitory effect on ASC proliferation (n = 5, *p < 0.05). (B) HGF
mRNA expression by human ASCs cultured with or without FGF-2 (10 ng/ml) on day 7. FGF-2induced upregulation of HGF mRNA was significantly inhibited by SP (n = 5, *p < 0.05), while
U1026 and SB had limited inhibitory effects. (C) Inhibitor specificity. Left: Phosphorylation of cJun increased following 15-minute treatment with FGF-2 (10 ng/ml). FGF-2-induced
phosphorylation of c-Jun was completely prevented by pretreatment with SP (n = 4, *p < 0.05).
Right: Phosphorylation of c-Jun increased with HGF treatment (10 or 100 ng/ml). HGF, even at ahigh concentration (100 ng/ml), did not increase c-Jun phosphorylation in human ASCs (n = 3). (D)Cell counts after 7 days in culture. FGF-2-induced cell proliferation was significantly inhibited by
the JNK inhibitor SP in all cell types examined (hASC, human adipose-derived stem/stromal cells;
hBM-MSC, bone marrow-derived mesenchymal stem cells; hDF, human dermal fibroblasts;
mASC, mouse adipose-derived stem/stromal cells; n = 5, *p < 0.05). (E) Real-time PCR assays for
HGF mRNA expression on day 7. HGF mRNA expression was promoted by FGF-2 in all cell
types, while the FGF-2-induced upregulation of HGF mRNA was significantly inhibited by a JNK
inhibitor except in human BM-MSCs (n = 5, *p < 0.05).

HGF secretion from ASCs induced by FGF-2

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HGF secretion from ASCs induced by FGF-2

Figure 4. Ischemia-reperfusion injury and subsequent regenerative changes in the inguinal adipose
tissue of mice.

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(A) Ischemia-reperfusion injury model. Top: The murine inguinal fat pad. The arrow indicates thedominant nutrient vessels arising from the femoral vessels. Arrowheads indicate communicating
branches to the skin. Bottom: The communicating branches were severed and the dominant vessels
were clamped for 3 hours with a vessel clip. (B) Hematoxylin-eosin staining revealed thatinterstitial infiltration of blood cells occurred as early as day 1 and diminished by day 7.
Regenerative changes were seen in the injured adipose tissue throughout the first week. Scale bars
= 100 µm. (C) Analysis of SVF by flow cytometry. Left: Total SVF cells and CD45+ cells, Right:
CD31–/CD34+ cells. Total SVF cells increased by day 7, with the similar increase of CD45+ cells,
while the number of CD34+/CD31– cells peaked on day 3 (n = 3, *p < 0.05). (D) Quantification of
BrdU-positive cells by immunohistochemistry (Supplemental online Fig. 2C). BrdU-positive
proliferating cells increased after injury, peaking on day 3 (n = 4, *p < 0.05). (E) Flow cytometricanalysis of SVF cells on day 3. BrdU-positive proliferating cells included both CD45+ and CD45–
cells; most BrdU+/CD45– cells (88.2 ± 4.2%, n = 3) were CD34+/CD31–. (F) Quantification of
TUNEL-positive cells by immunohistochemistry (Supplemental online Fig. 2D). TUNEL-positive
apoptotic cells were observed most frequently 1day after injury (n = 4, *p < 0.05). (G) Flowcytometric analysis of SVF cells for expression of Annexin V and CD45 on day 1. Most Annexin
V-positive apoptotic cells (90.1 ± 3.0%, n = 3) were CD45+. (H) Living tissue stained with
Hoechst 33342 (blue), lectin (endothelial cells; red), or PI (necrotic cells; green), imaged andmerged with interference contrast images. PI-positive necrotizing cells increased on day 3; some ofthese cells (17.2 ± 5.5%, n = 3) appeared to be lectin-positive capillary endothelial cells. Scale bars
= 100 µm.

HGF secretion from ASCs induced by FGF-2

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HGF secretion from ASCs induced by FGF-2

Figure 5. Expression of FGF-2 and HGF after ischemia-reperfusion injury to the adipose tissue.
(A) Immunostaining for FGF-2. FGF-2 protein was detected as early as 1 day post-injury with thepeak of detection on day 3. Scale bars = 100 µm. (B) Western blot analysis for FGF-2. FGF-2
protein increased significantly on days 1 and 3 (n = 3, *p < 0.05). (C) Immunostaining for HGF.
Little HGF protein was detected 1 day after injury, but it was detected as late as 3 days post-injury,
peaking 7 days after injury. Scale bars = 100 µm. (D) Western blot analysis for HGF. HGF proteinincreased significantly on day 7 (n = 3, *p < 0.05). (E) Real-time PCR assay of FGF-2 mRNA andHGF mRNA expressions in the injured adipose tissue. FGF-2 mRNA was downregulated soonafter injury (n = 5, *p < 0.05), while HGF mRNA was significantly upregulated 3 days after injury(n = 5, *p < 0.05).

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HGF secretion from ASCs induced by FGF-2

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HGF secretion from ASCs induced by FGF-2

Figure 6. Cellular events in the repair process of injured adipose tissue.

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(A) The injured tissue on day 3. Top: Staining for CD34 (green), BrdU (red) or Hoechst 33342(blue). Most BrdU-positive cells, frequently observed 3 days after injury, were also positive forCD34. Scale bars = 20 µm. Bottom: Living tissue image of the tissue on day 3 stained with CD34
(green), lectin (endothelial cells; red), or Hoechst 33342 (nuclei; blue).On day 3, most of CD34+
cells were lectin-negative, suggesting that they were ASCs. Scale bars = 20 µm. (B) Living tissue
image stained with BODIPY (adipocytes; yellow), lectin (endothelial cells; red), or Hoechst 33342(nuclei; blue). Increased number of nucleated cells and small-sized adipocytes were observed ondays 1 and 3. Lectin+ small round cells, which may be infiltrating blood-derived cells, were seen
on day 1, though they remained to be characterized. Large-sized adipocytes increased on day 7, but
not when treated with a JNK inhibitor. Blue scale bars = 200 µm, and white scale bars = 50 µm.
(C) Quantification of small adipocytes (less than 50 µm in diameter). In the group treated with a
JNK inhibitor, the percentages of small adipocytes were significantly higher on days 7 and 14 (n =3, *p < 0.05). (D) Three-dimensional images of living tissue stained with BODIPY (adipocytes;
yellow), PI (nuclei of necrotizing cells; red), or Hoechst 33342 (all nuclei; blue); images dissectedhorizontally and longitudinally at levels of white broken lines are also shown. PI-positive cells
were frequently observed on days 1 and 3; some of these cells (10.7 ± 5.7% on day 1; 4.4 ± 0.5%
on day 3; n = 3) were proven to be adipocytes. Treatment with a JNK inhibitor increased thenumber of PI-positive cells on day 7. Scale bars = 50 µm. (E) Quantification of PI-positive cells. In
the group treated with a JNK inhibitor, the percentage of PI-positive cells was significantly higher
on day 7 (n = 3, *p < 0.05).

HGF secretion from ASCs induced by FGF-2

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HGF secretion from ASCs induced by FGF-2

Figure 7. Influences of signal inhibition after ischemia-reperfusion injury to adipose tissue.
(A) Proliferating cell counts according to immunohistology on day 3. The injury-induced increasein BrdU-positive proliferating cells was significantly inhibited by a JNK inhibitor (SP) or an antiFGF-2 antibody (n = 4, *p < 0.05), but not by an anti-HGF antibody. (B) HGF mRNA expression
by adipose tissue on day 3. Continuous administration of SP or anti-FGF-2 antibody prevented
upregulation of HGF mRNA on day 3 (n = 5, *p < 0.05). (C) Immunostaining for HGF on day 7.
Continuous administration of SP or anti-FGF-2 antibody inhibited HGF secretion in the injured
adipose tissue on day 7. Scale bars = 100 µm. (D) Western blot analysis for HGF on day 7.
Treatment with SP and anti-FGF-2 antibody inhibited the expression of HGF protein. (E)Fibrogenesis in the injured adipose tissue at 2 weeks after injury. Fibrotic area was stained with
Azan staining. Scale bar = 1 mm. Ischemia-reperfusion injury induced significant fibrogenesis; thearea of fibrosis was twice as large as that in the uninjured animal. Continuous administration of SP,
anti-FGF-2, or anti-HGF antibody significantly increased fibrosis (n = 6, *p < 0.05).
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HGF secretion from ASCs induced by FGF-2

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IFATS Series: FGF-2-induced HGF Secretion By Adipose-Derived Stromal Cells
Inhibits Post-Injury Fibrogenesis Through A JNK-Dependent Mechanism
Hirotaka Suga, Hitomi Eto, Tomokuni Shigeura, Keita Inoue, Noriyuki Aoi, Harunosuke
Kato, Satoshi Nishimura, Ichiro Manabe, Koichi Gonda and Kotaro Yoshimura
Stem Cells published online Sep 4, 2008;
DOI: 10.1634/stemcells.2008-0261
This information is current as of September 4, 2008
Updated Information& Services

including high-resolution figures, can be found at:
http://www.StemCells.com

Supplementary Material

Supplementary material can be found at:
http://www.StemCells.com/cgi/content/full/2008-0261/DC1
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