A sex-specific role for androgens in angiogenesis

Abstract

Mounting evidence suggests that in men, serum levels of testosterone are negatively correlated to cardiovascular and all-cause mortality. We studied the role of androgens in angiogenesis, a process critical in cardiovascular repair/regeneration, in males and females. Androgen exposure augmented key angiogenic events in vitro. Strikingly, this occurred in male but not female endothelial cells (ECs). Androgen receptor (AR) antagonism or gene knockdown abrogated these effects in male ECs. Overexpression of AR in female ECs conferred androgen sensitivity with respect to angiogenesis. In vivo, castration dramatically reduced neovascularization of Matrigel plugs. Androgen treatment fully reversed this effect in male mice but had no effect in female mice. Furthermore, orchidectomy impaired blood-flow recovery from hindlimb ischemia, a finding rescued by androgen treatment. Our findings suggest that endogenous androgens modulate angiogenesis in a sex-dependent manner, with implications for the role of androgen replacement in men.

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J. Exp. Med. Vol. 207 No. 2 345-352
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345
Brief Definitive Report
The role of androgens in cardiovascular disease
(CVD) remains controversial. Although men de-
velop CVD earlier than women, consistent with
increased atherosclerosis (Wingard et al., 1983;
Shaw et al., 2006), repair/adaptation mecha-
nisms may be enhanced in males (Joseph and
Dyson, 1965). For example, male sex is associ-
ated with increased collateralization in patients
with severe coronary artery lesions (Abaci et al.,
1999). Androgens have been implicated in play-
ing a detrimental role in CVD by numerous in
vitro studies (McCrohon et al., 1999; Ng et al.,
2003a,b), but in vivo and clinical studies fail to
support this relationship (Liu et al., 2003; Wu
and von Eckardstein, 2003; Harman, 2005).
Indeed, some but not all cohort studies suggest
that subnormal blood levels of testosterone are
deleterious and correlate with increased cardio-
vascular risk factors and mortality (Araujo et al.,
2007; Khaw et al., 2007; Laughlin et al., 2008;
Tivesten et al., 2009).
Angiogenesis is a prerequisite for embryonic
development and plays a critical role in adult
physiological processes such as wound repair
and in tissue responses to ischemia (Carmeliet,
2005). The cardiovascular system is an important
target of androgen action (Liu et al., 2003), and
although the eect of estrogen in angiogenesis
has been studied extensively (Losordo and Isner,
2001), the role of androgens in this process
remains largely unexplored.
Androgens have been implicated in the re-
newal of various tissues, including muscle and
bone (Harman, 2005), but their potential role
in the maintenance of cardiovascular repair
mechanisms is unknown. As androgens have been
implicated in the regulation of angiogenesis-
related genes (Ng et al., 2003b; Death et al.,
2004), we reasoned that androgens may play a
role in angiogenesis, a process critical in the re-
generative response after ischemia. To examine
the role of androgens in angiogenesis, we used
dihydrotestosterone (DHT), the most potent
natural androgen that acts via the androgen
receptor (AR). As testosterone action partly
depends on its conversion to estradiol by the
unique enzyme CYP19 (aromatase), whereas
CORRESPONDENCE
Martin K.C. Ng:
mkcng@med.usyd.edu.au
OR
Daniel P. Sieveking:
sievekingd@hri.org.au
Abbreviations used: AcLDL,
Dil-acetylated low density
lipoprotein; ANOVA, analysis
of variance; AR, androgen
receptor; CVD, cardiovascular
disease; DHT, dihydrotestos-
terone; EC, endothelial cell;
ER, estrogen receptor; HF,
hydroxyutamide; HIF-1,
hypoxia-inducible factor 1;
LDPI, laser Doppler perfusion
index; MNC, mononuclear
cell; mRNA, messenger
RNA; PI3K, phosphoinositol
3-kinase; SDF-1, stromal
cell–derived factor 1;
siRNA, small interfering
RNA; VEGF, vascular endo-
thelial growth factor; vWF,
von Willebrand factor.
A sex-specic role for androgens
in angiogenesis
Daniel P. Sieveking,1,2 Patrick Lim,1 Renée W.Y. Chow,1 Louise L. Dunn,1
Shisan Bao,3 Kristine C.Y. McGrath,1 Alison K. Heather,1
David J. Handelsman,4,5 David S. Celermajer,1,2,6 and Martin K.C. Ng1,2,6
1Heart Research Institute, Sydney 2042, Australia
2Department of Medicine and 3Department of Pathology, University of Sydney, Sydney 2050, Australia
4ANZAC Research Institute, Sydney 2139, Australia
5Department of Andrology, Concord Hospital, Sydney 2139, Australia
6Department of Cardiology, Royal Prince Alfred Hospital, Sydney 2050, Australia
Mounting evidence suggests that in men, serum levels of testosterone are negatively cor-
related to cardiovascular and all-cause mortality. We studied the role of androgens in
angiogenesis, a process critical in cardiovascular repair/regeneration, in males and females.
Androgen exposure augmented key angiogenic events in vitro. Strikingly, this occurred in
male but not female endothelial cells (ECs). Androgen receptor (AR) antagonism or gene
knockdown abrogated these effects in male ECs. Overexpression of AR in female ECs con-
ferred androgen sensitivity with respect to angiogenesis. In vivo, castration dramatically
reduced neovascularization of Matrigel plugs. Androgen treatment fully reversed this effect
in male mice but had no effect in female mice. Furthermore, orchidectomy impaired blood-
ow recovery from hindlimb ischemia, a nding rescued by androgen treatment. Our nd-
ings suggest that endogenous androgens modulate angiogenesis in a sex-dependent
manner, with implications for the role of androgen replacement in men.
© 2010 Sieveking et al. This article is distributed under the terms of an Attribu-
tion–Noncommercial–Share Alike–No Mirror Sites license for the rst six months
after the publication date (see http://www.jem.org/misc/terms.shtml). After six
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mercial–Share Alike 3.0 Unported license, as described at http://creativecommons
.org/licenses/by-nc-sa/3.0/).
The Journal of Experimental Medicine

346 Androgens and angiogenesis | Sieveking et al.
Chronic exposure to DHT (72 h) induced a dose-dependent
increase in male EC tubulogenesis (Fig. 1, D and E), as did 24 h
of exposure (Fig. S1). Similarly, DHT augmented male EC
proliferation in a dose-dependent fashion after 48 h (Fig. 1 F).
Again, addition of HF abolished all androgen-mediated ef-
fects on these key angiogenic events, consistent with an AR-
dependent mechanism (Fig. 1, B, C, E, and F).
As an independent method to verify the pivotal involve-
ment of AR in DHT-mediated angiogenic eects, we also
assessed tubulogenesis in male ECs subjected to small inter-
fering RNA (siRNA)–mediated knockdown of AR. DHT
eects were also abolished in ECs exposed to AR siRNA (Fig.
1 G). DHT can also mediate eects via direct cross-reactivity
at the ER at suciently high concentrations. Therefore, to
further exclude any contribution to DHT eects via eects
on ERs, we assessed tubulogenesis in male ECs treated with
the highest concentration of DHT with or without the ER
antagonist ICI182780. The addition of ICI182780 did not
have any eect on DHT-mediated augmentation of tubulo-
genesis (Fig. 1 H). These ndings are consistent with an AR-
mediated angiogenic eect in male ECs.
Androgens do not augment angiogenic events in vitro
in female ECs
Mounting evidence indicates that sex steroids regulate vari-
ous vascular biological processes in a sex-dependent fashion
DHT cannot be aromatized, the use of DHT avoids the
potentially confounding estrogen receptor (ER)–mediated
eects of testosterone.
RESULTS AND DISCUSSION
Androgens augment angiogenic events in vitro
in male endothelial cells (ECs)
Male ECs treated with DHT demonstrated a dose-dependent
increase in key angiogenic processes in vitro (Fig. 1). EC mi-
gration was assessed by two dierent methods. Cells were pre-
treated with varying doses of DHT (0, 4, 40, and 400 nM) and
subsequently assessed using a Boyden chamber assay or a
scratch wounding assay. Using the Boyden chamber assay, 24 h
of DHT administration induced dose-dependent male EC
migration (220 ± 22, 262 ± 38, and 336 ± 39% vs. 100% con-
trol value for DHT at 4, 40, and 400 nM, respectively; P <
0.001 using analysis of variance [ANOVA]; Fig. 1 B). Similar
results were obtained using a scratch wounding assay. Migra-
tion by male ECs was augmented in a dose-dependent fashion
(138 ± 7, 280 ± 23, and 306 ± 32%; P < 0.01 using ANOVA;
Fig. 1 C). Interestingly, in both assays, addition of the AR
antagonist hydroxyutamide (HF) abrogated DHT-mediated
EC migration (96 ± 6 and 94 ± 6% vs. 100% control value for
40 and 400 nM DHT + HF, respectively; P > 0.05; Fig. 1,
B and C). DHT eects on tubulogenesis were assessed using
both Matrigel and co-culture methods (Sieveking et al., 2008).
Figure 1. DHT augments key angiogenic events in vitro in male ECs via the AR. (A and B) Boyden chamber (cells were stained with Ulex lectin
[UEA-1] and DAPI) and (C) scratch wounding assays of migration by male ECs pretreated with DHT or vehicle (0.1% EtOH) for 24 h ± HF, assessed after 6
and 24 h, respectively. Bar, 50 µm. (D and E) Vascular network formation by male ECs exposed to DHT or vehicle ± HF for 72 h. Tubule area was quantied
using image analysis software (ImageJ; available at http://rsbweb.nih.gov/ij/). Bar, 100 µm. (F) Proliferation of male ECs exposed to DHT or vehicle ± HF for
24 h, assessed by direct cell counting (n = 4 independent experiments for A–F, respectively). (G and H) Matrigel assays of male ECs transfected with siRNA
targeted to the AR (G) or pretreated with the ER blocker ICI182780 and treated with DHT (H; n = 3 independent experiments). **, P < 0.01; and ***, P <
0.001 versus control using ANOVA. All data are expressed as means ± SEM. For each independent experiment, cells from a different donor were used.

JEM VOL. 207, February 15, 2010 347
Brief Definitive Report
for 0, 4, 40, and 400 nM DHT, respectively; P = 0.0024;
Fig. 3 A). Similarly, DHT induced dose-dependent increases
in the messenger RNA (mRNA) expression of VEGF re-
ceptors 1 and 2 (Flt-1 and KDR, respectively; Fig. 3, B and C).
However, ow cytometric assessment of surface VEGF recep-
tor levels revealed that DHT augmented KDR but not Flt-1
expression in a dose-dependent manner (Fig. 3, D and E). As
KDR is the chief mediator of the mitogenic/angiogenic action
of VEGF in ECs, whereas Flt-1 may function as a negative
regulator of VEGF action (Ferrara et al., 2003), our ndings
are consistent with a proangiogenic role for DHT through
VEGF signaling via KDR. Furthermore, the addition of anti-
VEGF antibody abrogated DHT-mediated increases in tubu-
logenesis (Fig. 3 F). Likewise, LY294002, an inhibitor of
phosphoinositol 3-kinase (PI3K), a key enzyme in the PI3K–
AKT pathway of VEGF signaling, inhibited DHT-mediated
tubulogenesis (Fig. 3 F). These ndings suggest that the proan-
giogenic eects of DHT in male ECs are VEGF dependent.
Endogenous androgens are necessary for angiogenesis
in vivo in males but not females
Having demonstrated a sex-specic role for androgens in
angiogenesis in vitro, we next assessed the role of androgens
in angiogenesis in vivo, rst using a Matrigel plug implanta-
tion model. Sexually mature male and female mice under-
went gonadectomy or a sham gonadectomy followed by
DHT administration. Orchidectomy markedly decreased
in vivo vascularization of Matrigel plugs (27.3 ± 2% mm2
von Willebrand factor [vWF]+ cells/mm2 Matrigel vs. con-
trol as 100%; P < 0.0001 using ANOVA; Fig. 4 A). As can
be observed in the representative image, these plugs were
virtually acellular. Androgen treatment fully reversed the cas-
tration-related impairment of angiogenesis in males, returning
(McCrohon et al., 1999; Ng et al., 2001; Ng et al., 2003b).
For androgens, this is related to an approximately two- to
fourfold increased AR expression in male vascular cells com-
pared with females (McCrohon et al., 2000; Death et al.,
2004). We therefore hypothesized that the proangiogenic
eects of DHT observed in ECs may also be sex dependent.
Unlike male ECs, exposure of female ECs to DHT had no
eect on migration (Fig. 2, A and B), tubulogenesis (Fig. 2 C),
or proliferation (P > 0.05; Fig. 2 D), consistent with andro-
gen-mediated sex-specic eects. To further examine the
role of the AR in mediating the sex-dependent eects of
DHT on angiogenesis, we overexpressed the AR in female
ECs (Fig. S2) and assessed tubulogenesis after exposure to
DHT. Female ECs overexpressing the AR became respon-
sive to DHT and displayed augmented tubulogenesis (114 ±
6% vs. 100% control value; P < 0.05 using the Student’s
t test), albeit to a lesser extent than in male cells (Fig. 2 E).
These ndings indicate that the sex-specic eects of DHT
on angiogenesis are at least in part mediated by sex dier-
ences in AR expression. As there are a variety of coregulators
involved in AR signaling (Liu et al., 2003), it is also possible
that many of these are also expressed in a sex-specic fashion
and are important for androgenic modulation of angiogenesis.
The proangiogenic effect of androgens in vitro are vascular
endothelial growth factor (VEGF) dependent
Next, to further elucidate the means by which DHT aug-
mented migration, proliferation, and tubulogenesis in male
ECs, cells exposed to DHT were assessed for the expression of
various proangiogenic factors. Exposure of male ECs to DHT
produced a dose-dependent increase in the production of
VEGF, a key angiogenic growth factor (102.8 ± 7, 107.8 ± 7,
118.9 ± 6, and 147.2 ± 15 pg VEGF/100 µg of cell protein
Figure 2. DHT does not augment key angiogenic events in vitro in female ECs. (A–D) Boyden chamber (A) and scratch wounding (B) assays of
migration assessed after 6 and 24 h, respectively, Matrigel assays (C), and proliferation by female ECs treated with DHT or vehicle (D; n = 4 indepen-
dent experiments). P > 0.05 using ANOVA. (E) Matrigel assays of female ECs transfected with an AR overexpression plasmid and treated with DHT (n =
3 independent experiments). *, P < 0.05 using the Student’s t test. All data are expressed as means ± SEM. For each independent experiment, cells from
a different donor were used.

348 Androgens and angiogenesis | Sieveking et al.
detectable as early as day 3 and signicant by day 5 after isch-
emia (LDPI at day 5 after ischemia: control, 0.28 ± 0.04; or-
chidectomized + DHT, 0.4 ± 0.05; P < 0.05 vs. orchidectomized
mice; Fig. 5 A). These ndings were also borne out in the
functional assessments of recovery (Fig. S3). The serial changes
in perfusion observed after femoral artery ligation have previ-
ously been shown to correlate with changes in vessel density
as determined by histological analysis (Counhal et al.,
1998). As early as day 7, immunohistochemical staining of
the adductor muscles of the ischemic hindlimbs revealed that
capillary density was impaired in orchidectomized mice and
augmented in DHT-treated orchidectomized mice (0.17 ±
0.01 and 0.27 ± 0.01 vs. 0.22 ± 0.01 capillaries/myober for
orchidectomized mice, orchidectomized + DHT mice, and
controls, respectively; P < 0.001 using ANOVA; Fig. 5, B
and C), consistent with the ndings observed for limb perfu-
sion. Mean vessel diameter was also signicantly smaller in
orchidectomized mice (P < 0.001; Fig. 5 D). Interestingly,
mean vessel diameter was also smaller in orchidectomized
mice receiving DHT, but DHT treatment appeared to miti-
gate the eects of castration. These data suggest that, in the
context of ischemia, endogenous androgens inuence the
rate and extent of ischemia-induced angiogenesis and play a
role in arteriogenesis.
To elucidate the mechanisms of androgen-induced angio-
genesis in the context of hindlimb ischemia, we assessed the
early expression of angiogenesis- and ischemia-related genes.
Notably, 3 d after ischemia, orchidectomy signicantly re-
duced the expression of hypoxia-inducible factor 1 (HIF-1;
0.5 ± 0.1 vs. 1.8 ± 0.2 relative mRNA expression ischemic/
nonischemic for orchidectomized mice vs. controls, respec-
tively; P < 0.05 using ANOVA) and stromal cell–derived
factor 1 (SDF-1; 0.7 ± 0.1 vs. 2.4 ± 0.1 relative mRNA
vascularization of Matrigel plugs to levels not signicantly
dierent to controls (91 ± 6% mm2 vWF+ cells/mm2 Matri-
gel vs. control as 100%; P < 0.001).
Consistent with previous ndings, ovariectomy attenuated
Matrigel plug vascularization in females (Morales et al., 1995),
albeit to levels more modest than in males (53 ± 8% mm2
vWF+ cells/mm2 Matrigel vs. control as 100%; P < 0.0001 us-
ing ANOVA; Fig. 4 B). In striking contrast to their male coun-
terparts, gonadectomy-related impairment of angiogenesis in
females was not reversed by DHT treatment (55 ± 6% vs. con-
trol as 100%; P < 0.0001 using ANOVA). In accordance with
our in vitro ndings, these data suggest that endogenous andro-
gens play a role in angiogenesis in males but not females.
Endogenous androgens modulate
ischemia-induced angiogenesis
Hypoxia is one of the most potent angiogenic stimuli (Ceradini
and Gurtner, 2005). To investigate the role of androgens in
ischemia-induced angiogenesis, we used a mouse model of
hindlimb ischemia involving the unilateral ligation and re-
moval of the femoral artery (Counhal et al., 1998). Male
mice were randomized to orchidectomy with or without
DHT treatment or a sham orchidectomy. Laser Doppler per-
fusion imaging demonstrated that orchidectomy markedly
inhibited the rate of recovery from hindlimb ischemia (laser
Doppler perfusion index [LDPI] ischemic/nonischemic ratio
after 11 d: control, 0.54 ± 0.04; castrate, 0.39 ± 0.04; P <
0.05; Fig. 5 A). In functional assessments of the ischemic
hindlimbs, orchidectomized animals also displayed impaired
recovery in foot movement and ischemic damage (Fig. S3).
Interestingly, DHT not only rescued the orchidectomy-
induced impairment of blood ow recovery but also enhan-
ced recovery from ischemia, with improvements in flow
Figure 3. Proangiogenic effects of DHT in male ECs is VEGF dependent. (A) Production of VEGF by male ECs exposed to DHT for 48 h. Total cell
lysate protein was quantied and assayed via ELISA (n = 4 independent experiments). P = 0.002 using ANOVA for linear trend. (B) Expression of Flt-1
(P = 0.0128) and (C) KDR (P = 0.0295) mRNA and (D) Flt-1 and (E) KDR (P = 0.0005) protein in male ECs exposed to DHT for 48 h (using ANOVA for linear
trend). (F) Vascular network formation in male ECs treated with DHT with and without 1 µg/ml anti-VEGF antibody or 10 µM of the PI3K inhibitor
LY294002 (n = 3 independent experiments for B–F, respectively). ***, P < 0.001 compared with control using ANOVA. All data are expressed as means ±
SEM. For each independent experiment, cells from a different donor were used.

JEM VOL. 207, February 15, 2010 349
Brief Definitive Report
DHT, the increases observed for SDF-1 and KDR in DHT-
treated mice are also related to induction of HIF-1 (Ceradini
et al., 2004). There were trends toward a reduction in VEGF
expression in orchidectomized animals and an increase with
DHT treatment that were not statistically signicant. Never-
theless, given the critical role of HIF-1 in inducing VEGF-
related angiogenic events in ischemia and an increased expression
of KDR, our in vivo ndings are compatible with a role for
expression ischemic/nonischemic for orchidectomized mice
vs. controls, respectively; P < 0.05 using ANOVA), which are
key factors in the coordination of angiogenic gene expres-
sion and progenitor cell migration and homing, respectively
(Fig. 5, E and F, respectively; Ceradini et al., 2004). In stark
contrast, DHT treatment of orchidectomized mice augmented
expression of HIF-1 (3 ± 0.8; P < 0.01) and SDF-1 (3.6 ±
0.9; P < 0.01). These mice also demonstrated increased ex-
pression of KDR (3 ± 0.8 vs. 0.6 for orchidectomized + DHT
vs. controls, respectively; P < 0.01 using ANOVA; Fig. 5 G),
providing support for a role of DHT in VEGF signaling in
vivo. Furthermore, similar trends were observed for VEGF and
neuropillin-1 (Fig. S4). HIF-1 is the key subunit to HIF-1,
which is a critical, genome-wide transcription regulator re-
sponsible for oxygen homeostasis and responsive to hypoxic
stress (Semenza, 2002; Ceradini and Gurtner, 2005). It drives
the expression of >100 genes, including key genes associated
with angiogenesis such as VEGF and its receptors (Semenza,
2002). It is noteworthy that androgens have been associated
with HIF-1 expression in the prostate (Boddy et al., 2005).
Hence, it is possible that in addition to the direct eects of
Figure 4. Endogenous androgens play a role in angiogenesis in
males but not females. (A) Vascularization of Matrigel plugs harvested
from male mice after 14 d, with representative images (n = 8/group;
combined data from four independent experiments are shown). Sections
were immunohistochemically stained for vWF (3,3-diaminobenzidine
[DAB] + nickel, eosin). Bars, 100 µm. (B) Vascularization of Matrigel plugs
harvested from female mice, with representative images (n = 5–6/group;
combined data from three independent experiments are shown). Sections
were immunohistochemically stained for vWF (DAB, hematoxylin). Bars,
50 µm. ***, P < 0.001 versus control using ANOVA. All data are expressed
as means ± SEM.
Figure 5. Endogenous androgens modulate ischemia-induced an-
giogenesis. (A) LDPI. Limb perfusion ratio (ischemic/nonischemic) is pre-
sented with representative images (n = 8/group; combined data from four
independent experiments are shown). Differences between groups were
assessed by two-way ANOVA with Bonferroni correction (brackets at
right). *, P < 0.05; and **, P < 0.01 for comparisons between orchidecto-
mized and orchidectomized + DHT mice at individual time points. (B) Rep-
resentative images of neovascularization of ischemic hindlimbs at day 7.
Capillary density was measured in 8-µm frozen sections of the adductor
muscles via immunohistochemical staining for vWF (arrows). Bars, 100 µm.
(C) Capillary density expressed as the number of capillaries per myober
(eosin) and (D) mean vessel diameter (n = 6/group; combined data from
three independent experiments are shown). (E–G) Mechanisms of andro-
genic modulation of angiogenesis in hindlimb ischemia at day 3. Quantita-
tive RT-PCR for the expression of the angiogenesis- and ischemia-related
genes (E) HIF-1, (F) SDF-1, and (G) KDR (n = 6/group; combined data
from three independent experiments are shown). *, P < 0.05; **, P < 0.01;
and ***, P < 0.001 versus control using ANOVA. All data are expressed as
means ± SEM.

350 Androgens and angiogenesis | Sieveking et al.
castration-mediated decline in ulex+/AcLDL+ angiogenic cells
in both the spleen (17.8 ± 0.9 cells/200× eld; P > 0.05 using
ANOVA) and the bone marrow (31.7 ± 5 cells/200× eld;
P < 0.001 using ANOVA). SDF-1 is a key chemokine in the
migration and homing of bone marrow–derived progenitor
cells to sites of ischemia, and is itself a ligand for the receptor
CXCR4 (Jin et al., 2006); therefore, we also assessed the levels
of CXCR4+/Sca-1+ progenitor cells after induction of hindlimb
ischemia. Consistent with increased expression of SDF-1,
orchidectomized mice receiving DHT had signicantly ele-
vated numbers of CXCR4+/Sca-1+ cells present in the bone
marrow at day 3 after ischemia (0.8 ± 0.2% vs. 0.4 ± 0.07% of
MNCs for orchidectomized + DHT vs. controls, respectively;
P < 0.05 using ANOVA; Fig. 6 C). Trends similar to those
seen for ulex+/AcLDL+ cells were observed in the spleen for
CXCR4+/Sca-1+ cells (i.e., decrease with castration and increase
with DHT treatment), but these were not statistically signi-
cant (Fig. 6 D). The mobilization of cells from the bone mar-
row to the periphery is a highly coordinated, time-dependent
event. Therefore, because sampling took place at day 3, it is
possible that the signicantly elevated numbers of Sca-1+/
CXCR4+ cells in the bone marrow would not correspond to
higher levels in the periphery until a later time point. Overall,
our ndings suggest that androgens play a role in the prolifera-
tion and mobilization of circulating angiogenic cell populations
in the context of ischemia.
Finally, in light of the well-established eects of andro-
gens on erythropoiesis (Kennedy and Gilbertsen, 1957), we
assessed erythroid progenitor cell levels, as these cells have
been demonstrated to augment angiogenesis (Sasaki et al.,
2007). Gonadal status had no eect on the numbers of gran-
ulocyte/macrophage progenitors present in the bone mar-
row or the spleen (Fig. 6, E and F). However, consistent
VEGF in this context. Overall, these ndings suggest that
endogenous androgens play a role in the coordination of
ischemia-mediated angiogenesis by the regulation of key
angiogenesis-related genes, including HIF-1 and SDF-1.
Androgens modulate ischemia-induced
angiogenic/progenitor cell mobilization
It is now clear that the mobilization of various angiogenic/
progenitor cells from the bone marrow to sites of ischemia is
critical for neovascularization (Sieveking and Ng, 2009). Because
the spleen serves as a reservoir for peripheral stem/progenitor
cells (Heeschen et al., 2003), to further elucidate the mecha-
nisms of androgen-induced angiogenesis in hindlimb ischemia,
the early mobilization of angiogenic/progenitor cells was as-
sessed in both the bone marrow and the spleen. Mononuclear
cells (MNCs) staining positive for ulex lectin and uptake of Dil-
acetylated low density lipoprotein (AcLDL) represent a hetero-
geneous population of cells with angiogenic activity (Asahara
et al., 1997). Although the precise role of these cells in angiogen-
esis has been debated, these cells have been reported to aug-
ment angiogenesis in a paracrine fashion through the secretion
of various growth factors (Heil et al., 2004; Sieveking et al.,
2008). In females, estrogen has also been demonstrated to mo-
bilize bone marrow–derived angiogenic/progenitor cells that
participate in cardiovascular regeneration (Iwakura et al., 2006).
Similarly, the ndings of this study show that androgens modu-
late the mobilization of angiogenic cells. Orchidectomy mark-
edly decreased the numbers of ulex+/AcLDL+ cells in the bone
marrow (22.8 ± 1 vs. 13.4 ± 0.9 cells/200× eld for control vs.
orchidectomized mice, respectively; P < 0.001 using ANOVA;
Fig. 6 A) and the spleen (20.6 ± 0.8 vs. 15.2 ± 0.7 cells/200×
eld for control vs. orchidectomized mice, respectively; P <
0.001 using ANOVA; Fig. 6 B). DHT treatment rescued
Figure 6. Endogenous androgens modulate angiogenic/progenitor cell mobilization in response to ischemia. (A and B) Ulex+/AcLDL+ cells in the
bone marrow (A) and spleen (B). Cells were seeded at a density of 5 × 106 cells/mm2 and after 4 d were assessed for the ability to ingest 4 µg/ml AcLDL
(Invitrogen) and to bind 10 µg/ml FITC–UEA-1 (Sigma-Aldrich) via uorescence microscopy. (C and D) SCA-1+/CXCR4+ cells in the bone marrow (C) and
spleen (D). Hematopoietic progenitor cells were assessed using methylcellulose assays. (E and F) Granulocyte/macrophage CFUs in the bone marrow
(E) and spleen (F). (G and H) Erythroid CFUs in the bone marrow (G) and spleen (H; n = 6/group; combined data from three independent experiments
are shown). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 using ANOVA. All data are expressed as means ± SEM.

JEM VOL. 207, February 15, 2010 351
Brief Definitive Report
Real-time RT-PCR. Real-time RT-PCR was performed in triplicate using
iQ SYBR Green Supermix and the iCycler Real-Time PCR Detection System
(Bio-Rad Laboratories). For the animal studies, the relative expression (R) of
each target mRNA was calculated based on the Pfa equation (Pfa, 2001),
as follows: R = (Etarget)∆Cttarget/(Ereference)∆Ctreference, where ∆Ct = Ctischemic 
Ctnonischemic. The reference was -actin or 2-microglobuin mRNA.
Animal studies. All animal studies were performed under ethical approvals
of the Sydney South West Area Health Service Animal Welfare Commit-
tee. Male and female C57Bl6/J mice at 6 wk of age were randomized to or-
chidectomy, ovariectomy, or sham castration. 10 d later, castrated mice
received a 1-cm subdermal silastic implant lled with crystalline DHT.
Matrigel plug implantation model. Matrigel plug assays were performed
as described previously (Passaniti et al., 1992). Commercially available
Matrigel (BD) without phenol red was supplemented with 100 ng/ml of
basic broblast growth factor.
Hindlimb ischemia model. Hindlimb ischemia and subsequent perfusion
(Moor Instruments) and monitoring was performed as described previously
(Stabile et al., 2003).
Angiogenic/progenitor cell mobilization. For ow cytometric analysis
(FC500; Beckman Coulter), MNCs were stained with PE-conjugated anti-
bodies against SCA-1 (BioLegend) and FITC-conjugated antibodies against
CXCR4 (BD) with matched isotype controls. CFU assays for granulocyte/
macrophage and erythroid progenitors were set up in methylcellulose me-
dium (MethoCult; STEMCELL Technologies Inc.).
Statistical analysis. Data are expressed as means ± SEM. Comparisons be-
tween groups were performed using a two-tailed t test or by a one-way
ANOVA with post hoc analyses for pairwise comparisons (Newman-Keuls
multiple comparison). Prism software (version 4.00; GraphPad Software, Inc.)
for Windows was used for statistical analysis.
Online supplemental material. Fig. S1 depicts a dose-dependent aug-
mentation of Matrigel-based tubulogenesis in male ECs exposed to DHT for
24 h. In Fig. S2, we show dierential gene expression of the AR in male and
female ECs as well as female ECs transfected with an AR expression vector.
Fig. S3 demonstrates the eect of castration and DHT treatment on the
functional recovery of male mice subjected to femoral artery ligation. Fig. S4
shows the expression of angiogenesis-related genes in response to hindlimb
ischemia. Online supplemental material is available at http://www.jem
.org/cgi/content/full/jem.20091924/DC1.
We wish to thank Professor R.H. Karas for providing scientic advice during the
preparation of this paper.
This study was supported by a grant from the National Health and Medical
Research Council (457534). D.P. Sieveking was supported by a postgraduate support
grant from GlaxoSmithKline Australia.
The authors have no conicting nancial interests.
Submitted: 3 September 2009
Accepted: 21 December 2009
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