The Epidermal Growth Factor-like Domain of CD93 Is a Potent Angiogenic Factor
et al. (2012) The Epidermal Growth Factor-like Domain of CD93 Is a Potent Angiogenic Factor. PLoS
ONE 7(12): e51647. doi:10.1371/journal.pone.0051647
The Epidermal Growth Factor-like Domain of CD93 Is a Potent Angiogenic Factor
Yuan-Chung Kao 0
Shinn-Jong Jiang 0
Wen-An Pan 0
Kuan-Chieh Wang 0
Po-Ku Chen 0
Ju Wei 0
Wei-Sheng Chen 0
Bi-Ing Chang 0
Guey-Yueh Shi 0
Hua-Lin Wu 0
Ken Arai, Massachusetts General Hospital/Harvard Medical School, United States of America
0 1 Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University , Tainan , Taiwan , 2 Cardiovascular Research Center, National Cheng Kung University , Tainan, Taiwan, 3 Center for Bioscience and Biotechnology , National Cheng Kung University , Tainan , Taiwan
Human CD93, an epidermal growth factor (EGF)-like domain containing transmembrane protein, is predominantly expressed in the vascular endothelium. Studies have shown that AA4, the homolog of CD93 in mice, may mediate cell migration and angiogenesis in endothelial cells. Soluble CD93 has been detected in the plasma of healthy individuals. However, the role of soluble CD93 in the endothelium remains unclear. Recombinant soluble CD93 proteins with EGF-like domains (rCD93D123, with domains 1, 2, and 3; and rCD93D23, with domains 2 and 3) were generated to determine their functions in angiogenesis. We found that rCD93D23 was more potent than rCD93D123 in stimulating the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Production of matrix-metalloproteinase 2 increased after the HUVECs were treated with rCD93D23. Further, in a tube formation assay, rCD93D23 induced cell differentiation of HUVECs through phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase and extracellular signal-regulated kinases-1/2 signaling. Moreover, rCD93D23 promoted blood vessel formation in a Matrigel-plug assay and an oxygen-induced retinopathy model in vivo. Our findings suggest that the soluble EGF-like domain containing CD93 protein is a novel angiogenic factor acting on the endothelium.
Funding: This work was supported by the National Science Council grant NSC99-2323-B-006-003 and a grant from the Aim for the Top University Plan of the
National Cheng Kung University through the Ministry of Education, Taiwan. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Angiogenesis, which involves the formation of new blood vessels
from pre-existing vessels, is a complex process that plays an
important role in various physiological and pathological
conditions, including embryonic development , wound healing ,
and tumor growth . During angiogenesis, angiogenic factors
activate and bind to the receptors on blood vessels. Subsequently,
the activated endothelial cells secrete matrix-metalloproteinases
(MMPs) to degrade extra-cellular matrix to allow pre-existing
vessels to proliferate and sprout to neighboring vessels. The
sprouting neo-vessels weave a connecting vessel web by tube
formation . In addition, activations of focal adhesion kinase
(FAK), extracellular signal-regulated kinases-1/2 (ERK1/2), and
phosphoinositide 3-kinase (PI3K)/Akt/endothelial nitric oxide
synthase (eNOS) axes are responsible for the cell migration,
proliferation, permeability, and homeostasis of the endothelium
CD93 is composed of 652 amino acids and belongs to type-I
transmembrane glycoprotein . The structure of human CD93 is
consisted of five distinct domains, including a unique C-type
lectin-like domain (CTLD) (designated as D1), a tandem array of
five epidermal growth factor (EGF)-like repeats (designated as D2),
a Ser/Thr rich mucin-like domain (designated as D3), a 25-amino
acid transmembrane domain (designated as D4), and a 47-amino
acid cytoplasmic domain (designated as D5) . Although the
predicted molecular mass of CD93 is 68 kDa, its relative migration
in SDS-PAGE under reducing conditions is 126 kDa, suggesting
that it is heavily glycosylated . Consistent with this notion,
further study has shown that the cell surface expression of CD93 is
stabilized by O-glycosylation . CD93 is selectively expressed on
myeloid cells (granulocytes and monocytes), platelets, stem cells,
and mainly in endothelial cells [8,9,10,11,12]. Subsequent studies
have demonstrated that in both humans and mice, CD93 is not
present on tissue macrophages and that the predominant site of
expression is the vascular endothelium [9,13,14]. In addition, it
has been reported that during embryogenesis, mRNA and protein
expression of mouse CD93 (also known as AA4) are first detected
in the endocardium and the vascular endothelium in day 9
embryos. At later stages of development, the expression pattern of
mouse CD93 is maintained by the formation of the capillary
network invading various organ rudiments, suggesting that CD93
is involved in angiogenesis but not vasculogenesis .
Several soluble and cell surface-bound mediators including
growth factors, cytokines, chemokines, proteolytic
matrix-degrading enzymes, and cell adhesion molecules have been documented
as angiogenic factors, which may promote neovascularization
[15,16]. Most of these angiogenic factors contain the EGF-like
domain, which may display mitogenic effect on endothelial cells
. Previous reports have shown that the soluble CD93
fragments containing the EGF-like domain is released during
inflammation in a peritonitis mouse model . Moreover,
elevated CD93 expression is also detected in the plasma of the
patients who have overcome from coronary artery disease .
Theses clinical observations imply a clue to further investigate the
impact of soluble CD93 on angiogenesis. However, whether
soluble CD93 functions as an angiogenic factor and which domain
is essential in angiogenesis are never addressed. In this study, we
generated recombinant CD93 (rCD93) domain proteins, including
domain 1 (rCD93D1), domains 2 and 3 (rCD93D23), and
domains 1, 2, and 3 (rCD93D123), to examine the effect of these
recombinant proteins on angiogenesis. The present study shows
that the EGF-like domain of CD93 induces endothelial cell
proliferation, migration, and signaling pathways in vitro, and
stimulates angiogenesis in vivo.
The Expression and Purification of rCD93 Domain
The rCD93D1, rCD93D23, and rCD93D123 (Fig. 1A) were
prepared by a mammalian protein expression system. The purified
rCD93D1, rCD93D23, and rCD93D123 were subjected to
SDSPAGE and assessed by western blotting with a monoclonal
anti-cMyc antibody (Ab). The theoretical molecular masses of rCD93D1
(20,346.76 Da), rCD93D23 (36,739.07 Da), and rCD93D123
(61,876.55 Da) were analyzed by the integrated bioinformatics
database ExPASy, respectively. However, the molecular masses of
rCD93D1, rCD93D23, and rCD931D23, as shown on
SDSPAGE (Fig. 1B), were 20, 71, and 95 kDa, respectively, which
suggested that they were heavily glycosylated, as they were in their
native state. The purified proteins were further identified by a
liquid chromatography mass spectrometry/mass spectrometry
(LC-MS/MS) (Fig. S1A).
Effect of rCD93 Domain Proteins on Human Umbilical
Vein Endothelial Cells (HUVECs) Proliferation
Effect of rCD93D23 and rCD93D123 on the HUVECs
proliferation was evaluated in the presence of 1% fetal bovine
serum (FBS) by a WST-1 assay and a cell counting manner. Both
rCD93D23 and rCD93D123 stimulated HUVEC proliferation
(Fig. 1, CE), while rCD93D1 showed a nonsignificant mitogenic
effect, suggesting that rCD93 containing an EGF-like domain
possessed mitogenic activity.
Effect of rCD93 Domain Proteins on HUVEC Migration
The chemotactic motility of HUVECs was assayed using
Transwell to investigate the effect of rCD93D23 and rCD93D123
on HUVEC migration. rCD93D23 and rCD93D123 induced
HUVEC migration in a dose-dependent manner, which reached
maximum stimulation at a concentration of 30 ng/ml (Fig. 2, A
and B). rCD93D1, however, had no effect on cell migration (data
not shown). Since rCD93D23 had obviously higher mitogenic
ability than rCD93D123 to stimulate cell proliferation (Fig. 1, C,
D, E) and migration (Fig. 2, A and B), it was specifically applied to
further experiments. When rCD93D23 was pre-mixed with
rCD93D23 Ab at a concentration of 5 mg/ml or was boiled for
30 min (Fig. 2C), the chemotactic response induced by rCD93D23
was attenuated to a level comparable to that of the control.
Moreover, rCD93D23 could induce expression and activation of
MMP-2 in a dose-dependent manner as shown by a gelatin
zymography assay (Fig. 2D). The results indicated that HUVEC
migration was specifically induced by rCD93D23.
Signaling Cascade Mediated by rCD93D23 in HUVECs
Since FAK, ERK1/2, and PI3K/Akt/eNOS pathways mediate
endothelial cell proliferation and migration during angiogenesis,
we next studied whether these signaling pathways were involved in
rCD93D23-stimulated HUVECs by analyzing the levels of
phospho- and total protein of the cell lysates with western blotting.
rCD93D23 could stimulate FAK, ERK1/2, Akt, and eNOS
phosphorylation in a dose-dependent manner (Fig. 3A).
Furthermore, rCD93D23 at 50 ng/ml was used to assess the
timedependent activation of signaling cascades in
rCD93D23-stimulated HUVECs. The degree of signal activation was altered with
the time of treatment (Fig. 3B). These data indicated that
rCD93D23, in addition to activating ERK1/2, also stimulated
the PI3K/Akt/eNOS pathway in HUVECs. To further examine
whether rCD93D23-stimulated HUVECs migration was through
the ERK1/2 and PI3K/Akt/eNOS pathways, cells were
pretreated with either 10 mM U0126 or 10 mM LY294002 for one hour
prior to being stimulated with rCD93D23. The stimulatory effect
of rCD93D23 on migration was blocked by U0126 or LY294002
(Fig. 3C). This result suggested that rCD93D23 mediated
HUVECs migration was predominantly through the FAK,
ERK1/2, and PI3K/Akt/eNOS pathways.
rCD93D23 Induces Vascular Tube Formation in vitro
The role of CD93 in angiogenesis was assessed by the
morphological differentiation of HUVECs on Matrigel.
Capillary-like structures formed complete networks after 6 h incubation
in the presence of different concentrations of rCD93D23 (Fig. 4A),
whereas only a small number of tubes were formed in the PBS
control. As Fig. 4A indicates, rCD93D23 markedly induced a
dose-dependent response of tube formation in HUVECs. The
stimulatory effect of rCD93D23 (50 ng/ml) on tube formation in
HUVECs was blocked by polyclonal anti-CD93 IgG (10 mg/ml),
10 mM U0126, and 10 mM LY294002 (Fig. 4B). The stimulatory
effect of tube formation induced by rCD93D23 disappeared when
the rCD93D23 was boiled (Fig. 4B). These results indicated that
rCD93D23 had a novel angiogenic activity mediated through the
ERK1/2 and PI3K/Akt/eNOS pathways.
rCD93D23 Promotes Angiogenesis in vivo
To further investigate the angiogenic activity of rCD93D23
in vivo, the effect of rCD93D23 on blood vessel formation was
examined by a murine angiogenesis assay and an
oxygeninduced retinopathy (OIR) assay. Matrigel can serve as a vehicle
for the slow release of angiogenic factors [19,20]. The results
showed that angiogenesis significantly increased in Matrigel
plugs containing 100 ng rCD93D23 or 50 ng vascular
endothelial growth factor (VEGF) (Fig. 5A). The degree of
angiogenesis was also determined by measuring the hemoglobin
(Hb) content of the recovered Matrigel plugs. As shown in
Fig. 5A, the Hb content of the Matrigel containing rCD93D23
or VEGF plus heparin had significantly increased compared to
that of the control. Moreover, blood vessels were apparently
observed in the plugs containing rCD93D23 or VEGF by
CD31 immunofluorescent staining (Fig. 5B). OIR is a
wellestablished model to investigate the temporal expression pattern
of angiogenesis in hyperoxia induced retinal vessel loss in the
neonatal mice . The results showed that administration of
rCD93D23 into the hyperoxia treated mice could significantly
induce faster retinal vessel regrowth and neovascularization than
subjected to SDS-PAGE (10%) under reducing conditions. Coomassie blue-stained purified rCD93D1 (lane 1), rCD93D23 (lane 2), and rCD93D123 (lane
3). Western blotting of purified rCD93D1, rCD93D23, and rCD93D123 with monoclonal mouse anti c-Myc Ab. HUVECs were incubated with different
concentrations of (C) rCD93D123 or (D) CD93D23, or 10 ng/ml of VEGF for 48 h. Subsequently, the proliferation of HUVECs was measured by WST-1
reagent at OD450. (E) The cell numbers were counted. Each value represents the mean 6 SD (n = 3), and similar results were obtained in three
independent experiments. **, p,0.01; ***, p,0.001 vs. medium alone.
the PBS control (Fig. 5C). These results suggested that
rCD93D23 could promote angiogenesis in vivo.
The EGF-like Domain is Essential for CD93 to Induce
We further tested whether rCD93D123 also has angiogenic
effect in the plug. rCD93D123-containing Matrigels were
subcutaneously injected into mice and the plugs at different
time points were harvested (days 1, 3, and 5). We extracted
total proteins in the plugs and performed a SDS-PAGE to
analyze the expression pattern of rCD93D123 by using c-Myc
monoclonal Ab, which recognized the c-Myc tag at the
Cterminus of rCD93D123. In comparison with the rCD93D123
protein control, the injected rCD93D123 in the Matrigel was
processed at day 1 after being injected into mice and the
amount of recovered protein decreased at day 3 and day 5 (Fig.
Figure 2. Effect of rCD93 domain proteins on the chemotactic migration and MMP secretion of HUVECs. A migration assay was
performed in Transwell. (A) HUVECs (1 6 105 cells) suspended in M199 supplemented with 1% FBS were added to the upper wells. The lower wells
were filled with different concentrations of rCD93D123 (A) or rCD93D23 (B), or with 10 ng/ml of VEGF. (C) The lower wells were filled with rCD93D23
(30 ng/ml) in the absence or presence of CD93D23 Ab (5 mg/ml), boiled rCD93D23, or 10 ng/ml of VEGF. Each value represents the mean 6 SD
(n = 3), and similar results were obtained in three independent experiments. *, p,0.05; **, p,0.01; ***, p,0.001 vs. medium alone. ###, p,0.001 vs.
rCD93D23. (D) The culture media of HUVECs treated with different concentrations of rCD93D23 or VEGF (10 ng/ml) were used in gelatin zymography.
The similar results were obtained in three independent experiments. ***, p,0.001 vs. medium alone.
Figure 3. rCD93D23 induces HUVEC migration through the activation of Akt and ERK. (A) rCD93D23 dose-dependently induced FAK,
ERK1/2, Akt, and eNOS phosphorylation. HUVECs were treated with different concentrations of rCD93D23 or VEGF (10 ng/ml) for 30 min. (B)
rCD93D23 time-dependently induced FAK, ERK1/2, Akt, and eNOS phosphorylation in HUVECs. HUVECs were stimulated with 50 ng/ml of rCD93D23
for various time periods as indicated. The similar results were obtained in three independent experiments. *, p,0.05; **, p,0.01; ***, p,0.001 vs.
control (C) HUVEC suspensions were pretreated with LY294002 (10 mM) or U0126 (10 mM). The lower wells were filled with rCD93D23 (30 ng/ml) or
VEGF (10 ng/ml). Each value represents the mean 6 SD (n = 3), and similar results were obtained in three different experiments. ***, p,0.001 vs.
control. ###, p,0.001 vs. rCD93D23.
Figure 4. rCD93D23 induces tube formation through the activation of Akt and ERK in HUVECs on Matrigel in vitro. (A) rCD93D23
induced tube formation in a dose-dependent manner. (B) The tube formation induced by boiled rCD93D23, and rCD93D23 in the presence of
polyclonal anti-CD93D23 IgG (10 mg/ml), U0126 (10 mM), or LY294002 (10 mM). Each value represents the mean 6 SD (n = 3), and similar results were
obtained in three independent experiments. **, p,0.01; ***, p,0.001 vs. control. ###, p,0.001 vs. rCD93D23.
S2A), while the Matrigel containing no rCD93D123 protein
revealed no positive band following the same extraction
procedure. These data indicate that rCD93D123 may be
processed into different length of fragments. Furthermore, a
murine angiogenesis assay and an OIR assay were performed to
test whether the EGF-like domain is essential for CD93 to
induce angiogenesis in vivo. Both rCD93D23 and rCD93D123
could significantly induce angiogenesis in Matrigel and
neovascularization in hyperoxia-treated retina and rCD93D23 had a
higher potency than rCD93D123. However, rCD93D1 could
not induce angiogenesis in vivo (Fig. S2, B and C). These data
rCD93D23 Activates EGF Receptor (EGFR)
Because rCD93D23 is an EGF-like domain-containing
recombinant protein, we tested whether rCD93D23 can bind to EGFR,
which is the key signaling transduction pathway in angiogenesis,
and examined the downstream ERK phosphorylation. Fig. S3
showed that rCD93D23 or rCD93D123, but not rCD93D1, could
bind to EGFR by a co-immunoprecipitation assay (Fig. S3A).
Furthermore, blockage of EGFR activity by 10 mM of AG1478
could significantly inhibit rCD93D23- or rCD93D123-induced
Figure 5. rCD93D23 stimulates angiogenesis in vivo. (A) FVB mice were each injected subcutaneously with Matrigel containing VEGF or
rCD93D23 near the abdominal midline. The Matrigel plugs were excised after 4 days and then photographed. The Hb content of the excised Matrigel
plugs was examined. Each value represents the mean 6 SD (n = 3), and similar results were obtained in three independent experiments. *, p,0.05 vs.
PBS; **, p,0.01 vs. PBS. (B) Immunofluorescent staining of Matrigel plugs. Green channel, CD31 staining indicated vessel architecture. Blue channel,
DAPI indicated nucleus. VEGF was used as a positive control. (C) The hyperoxia-induced retina vessel loss mice were intraperitoneally injected with
rCD93D23 (160 mg/kg) (retina numbers = 20) or PBS (retina numbers = 7) twice a day for 2 days. Subsequently, the mice were sacrificed to harvest the
retinas for isolectin staining. The vessel density was quantitated by Photoshop software and analyzed by two-tailed Students t test. The results were
pooled from three independent experiments, all of which had similar results. **, p,0.01 vs. PBS. The white arrows indicated the neovascularization
ERK phosphorylation (Fig. S3B). These data indicate that EGFR
could be one of the potential receptors on endothelial cells for
CD93 knockout mice have no abnormalities in vascular
development , suggesting that CD93 is not essential in
vasculogenesis and embryonic development. However, CD93 is
dominantly expressed in the endothelium , and soluble CD93
fragments were detected in the circulation of patients with
cardiovascular disease . The roles of soluble CD93 in
endothelium are not investigated. Our data first demonstrated,
for the first time, that rCD93D23 with the EGF-like domain is a
novel angiogenic factor. rCD93D23 can stimulate proliferation,
migration, and in vitro tube formation in HUVECs (Fig. 1, 2, and
4). It also activates PI3K/Akt/eNOS and EKR1/2 (Fig. 3)
signaling pathways that are involved in a number of cellular
functions, including cell survival, migration, and angiogenesis
[3,24]. Moreover, rCD93D23 can significantly induce
angiogenesis in vivo (Fig. 5). Our findings should encourage further
investigations into the roles of CD93 in angiogenesis-related
Previous studies showed that CD93 might contribute to
macrophage-mediated phagocytosis of apoptotic cells in vivo .
The CD93 ectodomain, with an N-terminal lectin-like domain and
EGF-like repeats, is susceptible to the MMP-mediated shedding
from the surface of human monocytes and neutrophils. Shedding
is induced by PKC activators such as phorbol dibutyrate and
phorbol myristate acetate [25,26]. These observations indicate
that soluble CD93 may participate in inflammation, especially in
the removal of damaged cells. Furthermore, various forms of
CD93 ectodomains were detected in the plasma of healthy human
donors, and incubation with proinflammatory cytokines such as
TNF-a or the lipopolysaccharide induced CD93 shedding from
monocytes . These findings suggest that monocytes may be the
source of soluble CD93s and that membrane-bound CD93 may be
shed in response to inflammation-associated mediators.
Shedding mechanisms have been described for several
transmembrane proteins that contribute to the process of angiogenesis.
Membrane-anchored forms of TGF-a, amphiregulin, and
heparin-binding EGF, which belongs to a prototypical form of the EGF
family, act as autocrine growth factors by activating the EGF
receptor . Adhesion molecules like E-selectin and vascular cell
adhesion molecule-1 can promote angiogenesis in their soluble
forms . Because CD93 fragments can be detected in human
plasma, it is likely that CD93 is also cleaved under physiological
conditions. Although the exact structure of soluble CD93 remains
unknown, soluble CD93s seem to include EGF-like domains (D2),
as observed from the results of sandwich ELISAs with monoclonal
Abs R139 and R3 . To this notion, we subcutaneously injected
rCD93D123-containing Matrigel into mice to check whether
rCD93D123 could be processed during angiogenesis. We found
that the injected rCD93D123 was processed at day 1 after being
injected into mice (Fig. S2A). However, in this assay we could only
detect c-Myc tagged fragments of CD93. These data indicate that
rCD93D123 may be processed into different length of fragments,
which may contain the EGF-like domain in the plugs.
Furthermore, these EGF-like domain-containing fragments can exhibit
angiogenic ability to locally trigger angiogenesis. These
observations are consistent with previous reports that the concentration of
EGF-like domain-containing CD93 fragments elevated in the
plasma of patients with coronary artery disease . It has been
reported that CD93 shedding was mediated by an MMP, but was
independent of the TNF-a-converting enzyme . Whether
MMP-mediated cleavage of CD93 functions in angiogenesis
through a paracrine or an autocrine mechanism remains to be
studied. Furthermore, whether these natural soluble CD93
subspecies mimic the mitogenic and/or angiogenic properties
observed for rCD93D23 is unknown. Our results demonstrate that
the EGF-like domain is essential for soluble CD93s to exhibit
proangiogenic effects on the endothelium. However, the possibility
that membrane-intercalated, intact CD93 exerts other biological
effects cannot be excluded.
EGFR plays an important role during angiogenesis, and it can
sense angiogenic factors stimulation to elicit downstream signaling
molecules activation . Increasing reports showed that some
EGF-like domains acted as EGFR agonists . In our
observations, we showed that rCD93D23 could induce FAK,
ERK, Akt, and eNOS phosphorylation in a dose-dependent and
time-dependent manner (Fig. 3, A and B). Since CD93 is also a
multiple EGF-like repeats containing glycoprotein, it makes us
speculate whether CD93 can induce angiogenesis through EGFR
activation. Consistent with this notion, we found that rCD93D23
or rCD93D123, but not rCD93D1, could bind to EGFR (Fig.
S3A) and activate downstream ERK phosphorylation (Fig. S3B).
In addition, our present data showed that rCD93D23 had higher
angiogenic activity than rCD93D123, including promotion of cell
proliferation and migration (Fig. 1 and 2) and stimulation of
angiogenic associated signaling (Fig. 3) in vitro. Furthermore, a
murine angiogenesis assay and an OIR assay were performed to
show that rCD93D1 could not induce angiogenesis in vivo (Fig. S2,
B and C). It implies that the CTLD of CD93 may have an
attenuating role on CD93-mediated angiogenesis. These
observations are consistent with our recent publication that CTLD of
recombinant thrombomodulin plays a suppressing role on
We demonstrated that soluble CD93s with an EGF-like domain
possessed angiogenesis-promoting activity (Fig. 5). Elevated soluble
CD93 may be a biomarker for coronary artery disease . We
found that rCD93D23 could significantly induce angiogenesis in a
murine Matrigel-plug assay (Fig. 5, A and B), suggesting that
soluble CD93s with an EGF-like domain have an important role in
eliciting angiogenesis. As shown in Fig. 5C, we observed that the
rCD93D23 not only promoted vessel re-growth but also induced
immature vessel tufts. Generally, the vessels generated by
neovascularization in retinopathy animal models are more leaky
and immature than the regrowth vessels . However, we have
not confirmed the role of soluble CD93s in physiological or
Angiogenesis contributes to metastasis  and the development
of inflammatory diseases such as rheumatoid arthritis [33,34].
Therefore, the therapeutic correction of angiogenesis, by
preventing disordered angiogenesis, is a potentially fruitful approach for
the treatment of a number of human diseases. Our study is the first
to report that CD93 domains can promote angiogenesis and that
they do so predominantly through the PI3K/Akt/eNOS and
ERK1/2 pathways. We suggest that further assessment of the
involvement of CD93 in pathophysiological angiogenesis be
conducted, and further studies on the potential of rCD93D23 as
a novel therapeutic agent for ischemia-related diseases are
Materials and Methods
Expression and Purification of rCD93 Domain Proteins
HUVECs cDNA was used as template in a PCR; a fragment
coding for rCD93D1 (residues Thr22 through Gly177) was
amplified by using D1 forward
(59_CCCAAGCTTGGGATGGCCACCTCCATGGGCCTGCTGC_39) and D1 reverse
(59_GCTCTAGAGCGCCTTTGAAGCTGAACTTGCACACG_39) primers; a fragment coding for CD93D23 (residues
Val258 through Lys580) was amplified by using D23 forward
(59_AATTCTAGATACTTTTGCCCGTCAGTGCCA_39) primers. The rCD93D123 fragment (residues
Thr22 through Lys580) was amplified by using rCD93D123
(59_CCCAAGCTTGGGATGGCCACCTCCATGGGCCTGCTGC_39) and D3 reverse primers. The
amplified DNA fragments were constructed into the pCR3 vector
(Invitrogen) with c-Myc as a tag for protein detection to generate
recombinant human rCD93D1, rCD93D23, and rCD93D123 in
the mammalian HEK293 expression system. Conditioned
medium containing secreted rCD93D1, rCD93D23, or rCD93D123
was applied to a nickel-chelating Sepharose column (Amersham
Pharmacia Biotech), and rCD93D1-, rCD93D23-, or
rCD93D123-containing fractions were collected by gradient
elution with 300 mM imidazole (Sigma-Aldrich). The endotoxin
of recombinant proteins were assessed by the Limulus Amoebocyte
Lysates assay, and the endotoxin levels were lower than 1.0 EU/
Characterization of rCD93 Domain Proteins
rCD93D1, rCD93D23, and rCD93D123 were verified by
LCMS/MS and the data was searched using MASCOT software
(Matrixscience) against the NCBI database (Fig. S1A). The
LCMS/MS data of rCD93D23 and rCD93D123 were generated by
using an electrospray ionization-Trap mass spectrometer
(ThermoFinnigan; LCQ DECA XP Plus) which was equipped with a
LC system (Perkin Elmer; Series 200). A C18 LC column
(15060.075 mm, 3.5 mm; Agilent, ZORBAX 300SB-C18) was
used. The extracted peptides were dissolved in an A buffer (H2O
with 0.01% formic acid). A fifty-minutes gradient was used,
ramping from 0% to 100% B buffer (100% acetonitrile with
0.01% formic acid) in three-linear gradient steps to elute peptides.
The raw data files were submitted to MASCOT (version 2.1;
Matrix Science) for protein identification. The error tolerance of
peptide mass and fragment mass was set to 61Da. The potential
glycosylation sites were predicted by NetOGlyC 3.1 software (Fig.
S1B). The sequencing informations of the overexpression vectors
(pCR3 vectors with domains 2 and 3, or domains 1, 2, and 3 of
HUVECs were purchased from Life Technologies Corporation.
The cells obtained through source facilities are consistent with the
applicable legal and ethical practices of the United States. The
cells from the second or third passages were used in all
Cell Proliferation Assay
To assess the ability of rCD93 domain proteins to stimulate
HUVECs proliferation, tetrazolium salt WST-1
(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate)
was used in accordance with the manufacturers instruction
(Roche). Briefly, HUVECs were treated with various
concentrations of rCD93D1, rCD93D23, rCD93D123, or VEGF (10 ng/
ml) (R&D Systems) for 48 h. WST-1 was added to each well, and
the absorbance was measured at a wavelength of 450 nm after
1.5 h of incubation. In addition, the actual cell numbers were also
manually counted to accurately show the effects of recombinant
proteins on cell proliferation.
Cell Migration Assay
HUVECs migration was measured using Transwell (Corning
Costar Corp.) with 6.5-mm-diameter polycarbonate filters (8-mM
pore size). The lower surface of each filter was coated with
500 mg/ml gelatin (Sigma-Aldrich). Different concentrations of
rCD93 domain proteins or 10 ng/ml VEGF were diluted in M199
containing 1% FBS, and the mixtures were placed in the lower
wells. HUVECs were seeded into each upper well at a density of
16105 in 100 ml of M199 medium containing 1% FBS and were
allowed to migrate for 4 h at 37uC. The cells were fixed with
methanol and stained with Giemsa (Sigma-Aldrich). The total
migrated cells were counted with an optical microscope
(Olympus). For the rCD93D23 functional test, rCD93D23 in the
absence or presence of 5 mg/ml mouse polyclonal anti-CD93D23
Ab, which was prepared in our laboratory from BALB/c mice
immunized with rCD93D23 protein was used to perform the
migration assay. To verify the role of ERK1/2 and PI3K/Akt in
rCD93D23-mediated cell migration, assays were performed in the
presence of U0126 (Promega), an MEK inhibitor, and LY294002
(Calbiochem), a PI3K inhibitor.
Assay of ERK1/2, Akt, eNOS, and FAK Phosphorylation
HUVECs were cultured to confluence in a 6-cm-diameter dish
and were incubated in M199 containing 1% FBS for 18 h. After
washing with PBS, cells were incubated with serum-free M199 for
6 h and were treated with the indicated concentrations of
rCD93D23. Cell lysates were separated by SDS-PAGE, and the
levels of phospho-ERK1/2 (Tyr-204) and total ERK1/2,
phospho-Akt (Ser-473) and total Akt, phospho-eNOS (Ser-1177)
and total eNOS, and phospho-FAK (Tyr-397) and total FAK were
analyzed by western blotting with specific antibodies (Cell
Signaling Technology). To verify the role of EGFR in
rCD93D23-mediated ERK activation, assay was performed in
the presence of AG1478 (Millipore), an EGFR inhibitor.
HUVECs were seeded on 10-cm-diameter dishes in
supplemented M199 medium. After 24 h, cells were rinsed twice with
serum-free M199 and were incubated in serum-free M199 with
various concentrations of rCD93D23 or VEGF (10 ng/ml) for
20 h. The conditioned medium containing 5 mg of secreted
proteins was analyzed by gelatin-based zymography . The
digested area appeared clear on a blue background, indicating the
location of MMPs activity.
In vitro Matrigel Angiogenesis Assay
After 16 h of serum and growth factor depletion, HUVECs
were resuspended at a density of 1.06105/ml in M199 containing
4% FBS. Cell suspensions (50 ml) treated with various
concentrations of rCD93D23, inhibitors, or VEGF were added to wells of
mslide Angiogenesis (Integrated BioDiagnostics), which contained
10 ml of growth factor-reduced Matrigel (BD Biosciences). Each
concentration was tested in quadruplicate in the same plate, and
capillary-like structures were photographed with Olympus camera
Murine Angiogenesis Assay
To analyze the angiogenic effect in vivo , growth
factorreduced liquid Matrigel (0.5 ml) was mixed with heparin (30 U/
ml) and either rCD93D1 (100 ng per plug), rCD93D23 (50 or
100 ng per plug), rCD93D123 (100 ng per plug), VEGF (50 ng
per plug), or PBS. The mixture was then subcutaneously injected
in FVB mice near the abdominal midline. Four days after
injection, mice were euthanized, and Matrigel plugs were
surgically removed. For macroscopic analysis of angiogenesis,
Hb content in Matrigel was measured with Drabkin reagent 525
(Sigma-Aldrich). For histological analysis, CD31 Ab (Abcam) was
used to detect blood vessels.
The 7-day-old (P7) C57BL/6 mice with nursing mothers were
subjected to hyperoxia (75% oxygen) for 5 days, which inhibited
retinal vessel growth and led to significant vessel loss. On P12, the
mice were returned to room air and intraperitoneally
administrated with rCD93D1, rCD93D23, rCD93D123 (160 mg/kg), or
PBS twice a day for 2 days. At P14, the mice were sacrificed to
harvest the retinas for further analysis. Both the re-growth vessels
and retinal neovascularization were stained with isolectin
(Invitrogen). The vessel density was quantitated by Photoshop software as
described previously .
Subconfluent HUVECs were starved in serum-free M199 for 4
hours, and then incubated with 0.5 mg of rCD93D1 (lane 1),
rCD93D23 (lane 2), or rCD93D123 (lane 3) for 30 minutes. The
associations of EGFR and recombinant proteins were tested by
immunoprecipitating EGFR with EGFR Ab (Santa Cruz
Biotechnology) and detecting recombinant proteins by
immunoblotting recombinant proteins with c-Myc Ab (Santa Cruz
Biotechnology). Rabbit IgG was used as a negative control (lane 4).
Animal care conditions and design of experiments were
approved by the Institutional Animal Care and Use Committee
of the National Cheng Kung University (Tainan, Taiwan).
Data are shown as mean 6 SD. Statistical significance was
analyzed by one-way ANOVA followed by Bonferroni post hoc
test. Two-tailed Students t test was applied to the OIR model
(GraphPad Prism version 5.0. GraphPad software). Differences
with p-value ,0.05 were considered significant.
Figure S1 Bioinformatics searches of rCD93D23 and
rCD93D123. (A) rCD93D23 and rCD93D123 after LC-MS/MS
analysis were identified by MASCOT software. (B) The potential
O-glycosylation sites of CD93 were predicted by NetOGlyC 3.1
software. (C) The sequencing informations of rCD93D23 and
rCD93D123 in pCR3 vectors were analyzed by Vector NTI
software. Yellow box, signaling peptide. Blue box, start codon.
Red box, stop codon.
Figure S2 The EGF-like domain is essential for CD93 to
induce angiogenesis. (A) The rCD93D123 (0.5 mg) -containing
Matrigel plugs were harvested at days 1, 3, and 5. The expression
pattern of rCD93D123 in plugs was identified by c-Myc
monoclonal Ab. M1, Matrigel only was used as a negative control
at day 1. D123, 100 ng of rCD93D123 was used to identify the
molecular weight of recombinant protein. (B) FVB mice were each
injected subcutaneously with Matrigel containing VEGF,
rCD93D1, rCD93D23, or rCD93D123 near the abdominal
midline. The Matrigel plugs were excised after 4 days and then
photographed. The Hb content of the excised Matrigel plugs was
examined. Each value represents the mean 6 SD (n = 3), and
similar results were obtained in three independent experiments. *,
p,0.05 vs. PBS; ***, p,0.001 vs. PBS. (C) The hyperoxia-induced
retina vessel loss mice were intraperitoneally injected with
rCD93D1, rCD93D23, or rCD93D123 (160 mg/kg) (retina
numbers = 4) or PBS (retina numbers = 3) twice a day for 2 days.
Subsequently, the mice were sacrificed to harvest the retinas for
isolectin staining. The vessel density was quantitated by Photoshop
software. *, p,0.05; **, p,0.01 vs. PBS. The white arrows
indicated the neovascularization tufts.
Figure S3 rCD93D23 activates EGFR. (A) rCD93D1,
rCD93D23, or rCD93D123 incubated HUVECs lysate were
immunoprecipitated with specific EGFR Ab, and then
immunoblotted by c-Myc Ab. (B) AG1478 effectively blocked
rCD93D23or rCD93D123-induced ERK phosphorylation. rhEGF,
recombinant human EGF, was used as a positive control.
Conceived and designed the experiments: YCK SJJ GYS HLW. Performed
the experiments: YCK SJJ WAP PKC HJW WSC. Analyzed the data:
YCK KCW PKC. Contributed reagents/materials/analysis tools: BIC.
Wrote the paper: YCK.
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