From the Age Dimension Research Center,
National Institute of Advanced Industrial Science and Technology
(AIST), Tsukuba, Ibaraki 305-8566, Japan and the ¶ Institute of
Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki
305-8572, Japan
Received for publication, July 22, 2002, and in revised form, October 30, 2002
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ABSTRACT |
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Mouse embryonic stem-derived cells were recently
shown to differentiate into endothelial and smooth muscle cells. In the
present study, we investigated whether human umbilical vein
endothelium-derived cells retain the potential to differentiate into
smooth muscle cells. Examination of biochemical markers, including
basic calponin, SM22 The progenitors of vascular endothelial cells
(ECs)1 and smooth muscle
cells (SMCs) have long been considered to be distinct from one another:
ECs originate from angioblasts or hemangioblasts (1), whereas SMCs
originate from neural crest and locally differentiating mesenchyme (2,
3). Fibroblast growth factors (FGFs) play key roles in the induction of
both angioblasts (4) and hemangioblasts (4, 5) from mesoderm, and most
endothelial precursors divide and differentiate in response to
vascular endothelial growth factor (6, 7). At odds with this general
view are several studies that suggest SMC-like cells can arise from
atrio-ventricular and dorsal aorta ECs (8-11). For instance,
transforming growth factor- Normal ECs of human origin require the presence of an endothelial cell
growth factor (e.g. FGF family members) in addition to serum
for growth in culture (13, 14). In the present study, we examined the
role of FGF in regulating the endothelial characteristics of human
umbilical vein endothelium-derived cells (HUVE-DCs), also widely known
as human umbilical vein ECs (HUVECs).
Materials--
HUVE-DCs were isolated using the previously
described method for isolating HUVECs (15). Human adult pulmonary
artery endothelial cells (HPAECs) and human adult pulmonary artery
smooth muscle cells (HPASMCs) were purchased from Kurabo Corp., Osaka,
Japan. Recombinant human TGF- Cell Culture--
HUVE-DCs and HPAECs were plated in type I
collagen-coated plastic culture dishes and maintained in M199 medium
(Sigma) supplemented with 15% fetal bovine serum (Filtron) with
or without 5 ng/ml FGF-1 plus 10 µg/ml heparin. The inclusion of
heparin was for optimum FGF-1 activity; heparin exerted no effects on
cell differentiation (data not shown). HPASMCs were cultured in
HuMedia-SG2 (Kurabo Corp., Osaka, Japan) medium supplemented with 5%
fetal calf serum, human epithelium growth factor (0.5 ng/ml), human FGF
(2 ng/ml), insulin (5 µg/ml), gentamycin (50 µg/ml), and
amphotericin B (50 ng/ml). For analysis of HPASMC contraction, cells
were transferred to FGF-1- and heparin-free M199 medium supplemented
with 15% fetal bovine serum and maintained for 2 weeks. After
this period, the cells were plated on collagen type I gels and
subjected to contraction analysis.
Immunocytochemical Study--
Cells were double-labeled with
polyclonal anti-vWF and monoclonal anti-calponin primary antibodies and
then secondarily labeled with Texas Red-conjugated donkey anti-rabbit
IgG and fluorescein isothiocyanate-conjugated sheep anti-mouse IgG,
respectively. The signals were then detected using a confocal laser
scanning microscope (Carl Zeiss, Jena, Germany).
Adenovirus Vectors--
A recombinant E1-deleted adenoviral
vector carrying mouse Smad7 cDNA ligated with
FLAG-epitope (18) under the control of cytomegalovirus promoters was
generated (AdCMV-Smad7) and purified as described previously
(19, 20).
Semiquantitative Analysis of mRNA
Expression--
Semiquantitative RT-PCR/Southern blot analysis was
carried out as described previously (21). Briefly, total RNA was
isolated from cells using Isogen (Nippon Gene), after which a 1-µg
sample was reverse-transcribed using M-MLV reverse transcriptase
(Invitrogen) according to the manufacturer's instructions. Using 4%
of the reverse-transcribed mix, cDNA fragments of test genes
were amplified within the linear range by PCR using AmpliTaq Gold
(PerkinElmer Applied Biosynthesis). The specific primers used are
listed in Table I. Aliquots of the PCR
products were resolved on 1.0% agarose gels and transferred to Hybond
N+ membranes (Amersham Biosciences). The membranes were then hybridized
with the corresponding cDNA probe labeled with digoxygenin-labeled
dUTP (DIG-High Prime, Roche Molecular Biochemicals). The signals were
visualized, incorporated, and quantitated using NIH Image software
(version 1.62).
Immunoblot Analysis--
The cells were lysed in lysis buffer
(1% Triton® X-100, 50 mM Tris-HCl (pH 8.0),
150 mM NaCl, 1× protease inhibitor mixture, CompleteTM (EDTA-free); Roche Molecular Biochemicals), and
the extracted proteins (60 µg) were resolved by SDS-PAGE and
electroblotted to polyvinylidene fluoride membranes (Millipore Corp.,
Bedford MA). The proteins of interest were then detected using
appropriate antibodies and an ECL Western blotting detection system
(Amersham Biosciences).
Cell Contraction Assay--
HUVE-DCs were first cultured with or
without FGF-1 plus heparin for 3 weeks on type I collagen-coated
plastic culture dishes, after which they were relaxed by incubation
with 10 mM 8-bromo-cGMP for 20 min at 37 °C and embedded
in collagen gels in 24-well plates (final concentration of 8-bromo-cGMP
in the gel was 1 mM) (2.4 × 105 cells in
0.75 ml gel/well). The collagen gels were prepared using Cellmatrix type I-P (0.3% porcine type I collagen;
Nitta-gelatin Corp., Osaka, Japan) following the manufacturer's
instructions. Briefly, 10× M199 medium cell suspension in 1× M199
medium, bicarbonate buffer, fetal bovine serum, and Cellmatrix type I-P
solution were combined at a ratio of 1:1:1:1.5:5.5, poured into wells,
and allowed to polymerize. The resultant SMC-embedded gels were covered
with 0.5 ml of M199 containing 15% fetal bovine serum. After
incubating the gels for 17 h, by which time they had detached from
the well bottoms, ET-1 (250 nM) was added to the culture
once every 30 min. In some cases, ET-receptor (ET-R) antagonists (type
A, BQ-123 (30 µM); type B, BQ-788 (10 µM))
were added to the culture for 30 min before application of ET-1. For
quantification, each gel piece was transferred into a 1.5-ml micro-tube
containing 500 µl of culture medium and then centrifuged, and its
volume was measured. For morphological observation, relaxed
differentiated cells were seeded onto precasted type I collagen gel
(400 µl in 24-well plates) at a density of 4 × 104
cells/well, cultured for 17 h, and then treated with ET-1 and photographed under a phase-contrast microscope.
To compare the contractile capacity of differentiated HUVE-DCs and
HPASMCs, relaxed cells were seeded onto precasted type I collagen gel
(150 µl in 48-well plates) at a density of 2.8 × 104 cells/well. After incubating for 6 h, the cells
were treated with 500 nM ET-1 for 25 min and then with 1 µM ET-1 for 5 min. Each gel was then carefully detached
from the well wall and bottom, incubated for an additional 30 min in
the same well, and then transferred onto a transparent plastic sheet.
The degree of cell contraction was then determined by optically
measuring the size of the cell sheets on the gel pieces.
Induction of SMC-like Cells from HUVE-DCs by Prolonged Deprivation
of FGF--
We initially established a primary culture of HUVECs
(HUVE-DCs) as described previously (15, 16). The cells were cultured in
M199 medium supplemented with 15% serum, FGF-1, and heparin in
collagen type I-coated flasks and were split 1:4 once every 3-4 days.
Cells were always used for experimentation before they had reached 16 population doublings. As shown in Fig.
1a (left), the
majority of cells appeared compact and uniform, and when cultured until
confluent, they assumed the "cobble stone" appearance typical of
ECs (not shown). Interestingly, when we removed FGF-1 and heparin from
the growth medium for 3 weeks, an enlarged, flat cell type emerged
(Fig. 1a, right). Immunostaining the cells for
EC- and SMC-specific markers, i.e. vWF (22-25) and basic
calponin (26-29), respectively, revealed that all cells maintained in
FGF-1-containing medium were positive for anti-vWF antibody
(red) and negative for anti-basic calponin (Fig.
1b, left), but a significant fraction of the
FGF-1-deprived cells (9.4 ± 3.0% in number; n = 8) were positive for anti-basic calponin antibody (green), a
marker of SMCs (Fig. 1b, right). We then examined
the expression of several SMC- and EC-specific marker genes in
FGF-deprived HUVE-DC cultures using RT-PCR combined with Southern blot
analysis (Fig. 1c). mRNA expression of all the SMC
marker genes examined, i.e. basic calponin, SM22
The above results suggest that HUVE-DC cultures are composed of a
heterogeneous population with respect to their potential to
differentiate into SMC-like cells. To minimize this heterogeneity and
exclude the possibility that SMCs may have contaminated the primary
HUVE-DC cultures, we carried out a limiting dilution of HUVE-DC
cultures in FGF-1-containing growth medium. After establishing 30 single-cell-derived populations, they were deprived of FGF. We found
that in all of the cloned cell populations, cells deprived of FGF were
initially positive only for vWF, but after a period of FGF deprivation,
some began to express basic calponin, confirming that SMC-like cells
can indeed differentiate from ECs (Fig.
2). We then classified the clones into
two groups: slowly differentiating and rapidly differentiating. Among
the slowly differentiating clones (e.g. CL1), which
represented 6 of the 30 cultures, basic calponin-positive cells were
not detected after 2 weeks of FGF deprivation (Fig. 2a,
left) but did appear within 3 weeks (not shown). Among the
rapidly differentiating clones (e.g. CL14), basic
calponin-positive cells appeared comparatively quickly (Fig. 2a, right). After 2 weeks, approximately
one-fifth of the cells (11-23%, as determined by immunostaining)
expressed basic calponin (Fig. 2b, middle;
open arrow). Most of the other cells (69-87%) expressed
vWF (left, arrowhead), and a small fraction
(0-8%) expressed both vWF and basic calponin (Fig. 2b,
right, closed arrows).
Consistent with the immunohistochemistry, semiquantitative analysis
using RT-PCR/Southern blot revealed that levels of basic calponin
mRNA reached a maximum in CL14 cells after 2 weeks of FGF
deprivation and in CL1 cells after 3 weeks of deprivation (Fig.
2c). Expression of another smooth muscle cell-specific
marker, SM22
To further investigate the characteristics of cells expressing SMC
markers, we next examined their responsiveness to ET-1, which is
produced by endothelium (33) and acts via a smooth muscle-specific
receptor (ET-R type A) to induce contraction. Although expression of
ET-R type A mRNA was undetectable in HUVE-DCs cultured with FGF
(Fig. 3a, week 0),
it was abundantly expressed after 2-4 weeks of FGF deprivation (Fig.
3a). We observed a similar induction of ET-R type A in CL1
cells (data not shown). Interestingly, the level of ET-R type A
expression in the rapidly differentiating CL14 cells was high even in
the presence of FGF (data not shown) and did not change during the
4-week period of FGF deprivation.
To determine whether they were capable of contraction, SMC-like cells
were induced by FGF-free medium for 3 weeks and then plated on collagen
type I gels containing 1 mM 8-Br-cGMP, which relaxed the
cells. After incubating the gels for 17 h, we examined the effects
of ET-1 (250 nM). Whereas the control cells appeared flat
and relaxed in the collagen gels (Fig. 3b, left),
ET-1 caused the cells to contract and become more rounded in shape
(Fig. 3b, middle). Measurement of the gel volumes
showed that ET-1 treatment reduced the volume of gels made up with
FGF-deprived cells to 79.9% of control (Fig. 3c,
D) but had no effect on gels made up with FGF-treated cells
(Fig. 3c, F). Pretreating the cells with specific
ET-R type A and type B antagonists (30 µM BQ123 and 10 µM BQ788, respectively) inhibited both the ET-1-evoked
change in morphology (Fig. 3b, right) and the gel
shrinkage (Fig. 3d). Thus, sensitivity to ET-1 and cell
contractility correlated with the emergence of SMC-like cells in culture.
To further characterize the HUVE-DC-derived SMC-like cells, their
contractility was compared with that of pure HPASMCs. Because of the
limited availability of the cells, contractility was analyzed by
comparing ET-1-evoked changes in cell area on collagen gels using
smaller numbers of cells than were used for the gel volume assay shown
in Fig. 3d. HUVE-DCs and CL14 cells cultured in FGF-free medium for 3 weeks or HPASMCs cultured in FGF-free medium were harvested and plated on type I collagen gels containing 0.5 mM 8-Br-cGMP. After incubating the cells for 6 h, the
areas occupied by cells treated (+) or not treated ( Activin A Signaling Is Activated in FGF-deprived HUVE-DCs and Is
Responsible for the SMC-like Differentiation--
Earlier studies
suggested that SMC-like cells arise from atrio-ventricular (8) and
dorsal aorta ECs (10) after stimulation with exogenous polypeptide
growth factors belonging to the TGF-
To examine the role of activin A signaling in the induction of SMC-like
differentiation, we first attempted to block the signaling by adding
excess exogenous follistatin to the cultures. HUVE-DCs were cultured
with or without FGF-1 for 5 days in the presence or absence of
follistatin (1 µg/ml applied every 12 h) (Fig. 4b). Adding follistatin to the culture medium significantly diminished induction of basic calponin and SM22
The importance of activin A signaling in the induction of SMC-like
differentiation of HUVE-DCs was examined further by comparing expression of activin A in CL1 and CL14 cells, which, respectively, show low and high potentials for SMC-like differentiation (Fig. 2).
Following FGF-1 deprivation, activin A expression rapidly increased in
CL14 cells, reaching 775% of control (Fig. 4e, right panel). By contrast, activin A expression in CL1 cells increased only modestly, reaching 152 and 176% of control after 4 and 8 days of
FGF-1 deprivation, respectively (Fig. 4e, right
panel). We also examined the effect of exogenously applied activin
A on cell differentiation and found that, in the absence of FGF,
exogenous activin A induced expression of basic calponin and SM22
We also attempted to block activin A signaling by overexpressing an
inhibitory Smad in HUVE-DCs. Because HUVE-DCs are difficult to
transfect using typical expression vectors, we infected the cells with
AdCMV-Smad7, an adenoviral vector carrying mouse Smad7. As
expected, the level of Smad7 expression was dependent on the vector
dosage (Fig. 5a). Transfection
with Smad7 significantly diminished positive Smad signaling in
HUVE-DCs: it inhibited both the activin A-induced phosphorylation of
Smad2 (Fig. 5b) and its nuclear translocation (38-41) (Fig.
5c). In addition, control cells, but not Smad7
transfectants, underwent SMC-like differentiation when FGF-deprived and
thus showed increased expression of basic calponin and SM22
Finally, because some TGF- We have shown that, although HUVE-DCs cultured with FGF-1
constitute a population of ECs, when deprived of FGF-1, a significant fraction will differentiate into cells that fulfill many of the criteria used to define SMCs and that Smad-dependent
activin A signaling is responsible for this differentiation. Because a
more detailed comparison of these differentiated HUVE-DCs and
SMCs obtained from the walls of the blood vessels is yet to be
conducted, we are tentatively designating these cells as SMC-like.
Still, this is the first study to clearly demonstrate that ECs obtained from a normal human vascular tube can differentiate into SMC-like cells
identified not only by biochemical markers but also by their ability to
contract in response to ET-1 and that SMC-like differentiation and
dedifferentiation are reciprocally controlled by activin A and
FGF-1.
It was notable that expression of activin A, follistatin, and Smad7 was
all up-regulated in the HUVE-DCs after FGF-1 deprivation and that the
up-regulation was reversed by administration of FGF-1. Since activin A
reportedly induces expression of Smad7 and follistatin mRNA (37,
42), we suggest that activin A signaling would tend to suppress
differentiation through a negative feedback mechanism involving
follistatin and Smad7, although obviously this effect is not sufficient
to completely inhibit activin A-induced differentiation of HUVE-DCs
into SMC-like cells when FGF-1 was not present. The signaling pathway
downstream of the FGF receptors involves tyrosine kinase-mediated
activation of the FGF receptor and subsequent phosphorylation of
various signaling molecules, including p38, phosphatidylinositol
3-kinase, and MEK (43-49). We are currently investigating which of
these mediators are important for inhibition of differentiation of
HUVE-DCs into SMC-like cells.
It is unclear whether it is appropriate to describe the induction of
SMC-like cells from HUVE-DCs as transdifferentiation. Although HUVE-DCs
maintained with FGF are widely studied as normal human endothelial
cells, their differentiated characteristics become better defined when
they are deprived of FGF for a short period; for example,
FGF-deprived HUVECs readily form tube-like structures on
extracellular matrix.2
Indeed, in the present study, removal of FGF transiently enhanced expression of vWF in cells that were negative for SMC markers. Thus,
HUVE-DCs cultured in the presence of FGF may be "immature" ECs with
the capacity to differentiate into "mature" ECs or to transdifferentiate into SMC-like cells. In this regard, our finding that a small fraction of the cells simultaneously expressed vWF and
basic calponin suggests that expression of endothelial- and smooth
muscle-specific antigens are at least not mutually exclusive.
It would also be interesting to know whether ECs derived from human
adult blood vessels harbor the same potential to differentiate into
SMC-like cells as do HUVE-DCs. We therefore examined the potential of
HPAECs to differentiate into SMC-like cells and found that, indeed,
HPAECs cultured for 1-3 weeks in the absence of FGF-1 showed increased
expression of mRNAs for prostaglandin E synthase, basic calponin,
and SM22 Also necessary is a more detailed characterization of the SMC-like
cells differentiated from HUVE-DCs. Although we found that differentiated HUVE-DCs were less responsive to ET-1 than HPASMCs, our
findings may simply reflect the fact that only a small proportion of
these cells were SMC-like; among rapidly differentiating HUVE-DCs, including CL14 cells, approximately one-fifth (11-23%) expressed basic calponin after 2 weeks of FGF deprivation. Alternatively, it is
also possible that, at the single cell level, SMC-like cells derived
from HUVE-DCs are not truly SMCs. A line of study addressing this
question is currently under way.
, prostaglandin E synthase, von Willebrand
factor, and PECAM-1, as well as cell contractility, showed that
whereas endothelium-derived cells cultured with fibroblast
growth factor can be characterized as endothelial cells, when deprived
of fibroblast growth factor, a significant fraction differentiates into
smooth muscle-like cells. Reapplication of fibroblast growth factor
reversed this differentiation. Activin A was up-regulated in fibroblast
growth factor-deprived, endothelium-derived cells; moreover, the
inhibitory effects of exogenous follistatin and overexpressed Smad7 on
smooth muscle-like differentiation confirmed that the differentiation was driven by activin A signaling. These findings indicate that when
deprived of fibroblast growth factor, human umbilical vein endothelium-derived cells are capable of differentiating into smooth
muscle-like cells through activin A-induced, Smad-dependent signaling, and that maintenance of the endothelial cell phenotype and
differentiation into smooth muscle-like cells are reciprocally controlled by fibroblast growth factor-1 and activin A.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (TGF-
1) induced expression of smooth
muscle
-actin (SM-
Actin) in bovine aortic ECs (8) and in
mesenchymal cells (10). Furthermore, one recent study suggested that
both ECs and SMCs develop from the same precursor: Flk1-expressing
cells derived from embryonic stem cells (12). In this case,
vascular endothelial growth factor promotes EC differentiation,
whereas platelet-derived growth factor-BB promotes mural cell differentiation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1, activin A, bone morphogenetic
protein (BMP)-4, and follistatin were from R&D Systems, Inc.
(Minneapolis, MN). BQ123, BQ788, 8-bromo-cGMP, and endothelin-1
(ET-1) were from Calbiochem. Recombinant FGF-1 was expressed using the
pET-3c system in BL21(DE3)pLysS cells and purified to homogeneity using heparin-Sepharose affinity chromatography as described previously (16).
Heparin and anti-calponin monoclonal antibody (clone hCP) was from
Sigma. Anti-von Willebrand factor (vWF) polyclonal antibody was
from Dako. Anti-FLAG monoclonal antibody was from Eastman Kodak Co.
Texas-Red-conjugated donkey anti-rabbit IgG and fluorescein isothiocyanate-conjugated sheep anti-mouse IgG were from Amersham Biosciences. Monoclonal anti-TGF-
1-, -
2-, -
3-neutralizing
antibody was from Genzyme. Anti-Smad2 monoclonal antibody was
from BD Transduction Laboratories. Antiserum PS2 recognizing the
phosphorylated C-tail of Smad2 was generated, and its specificity was
tested as described previously (17).
Sequences of primers used in RT-PCR
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(30,
31), and prostaglandin E synthase (32), was clearly enhanced; indeed,
expression of basic calponin and SM22
increased up to 50-fold during
the 4-week period of FGF deprivation. Notably, expression of vWF
mRNA was also increased in FGF-deprived cells after 1 week but had
returned to its original level within 4 week. On the other hand,
expression of another endothelial-specific gene, pecam-1
(12), was little changed.
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Fig. 1.
HUVE-DCs express SMC
markers after prolonged FGF-1 deprivation. In a and
b, HUVE-DCs were grown in medium 199 containing 15% fetal
bovine serum with (+) FGF-1 (5 ng/ml) and heparin (10 µg/ml).
For induction of differentiation, the FGF-1 and heparin were removed
( ) from the medium, and the cells were cultured for 3 weeks. In
a, cells were photographed under a phase-contrast
microscope. In b, cells were immunostained for vWF
(red) and basic calponin (green). The scale
bar = 50 µm. In c, HUVE-DCs were cultured for
the indicated periods in the absence of FGF-1 and heparin, after which
expression of mRNAs encoding various SMC and EC markers was
examined by semiquantitative RT-PCR/Southern blot analyses (left
panels). The relative expression level of each mRNA was
plotted after normalization with GAPDH expression (right
panels). For SMC markers, the maximal level detected was defined
as 100% expression; for EC markers, the initial level was defined as
100% expression.
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Fig. 2.
Cloned HUVE-DCs retain the potential to
differentiate into and revert from SMC-like cells. HUVE-DCs were
cloned by limiting dilution; shown are representative clones. CL1 is a
slowly differentiating clone, and CL14 is a rapidly differentiating
one. In a, the cells were cultured for 2 weeks in the
absence of FGF-1 and heparin and immunostained for vWF (red)
and basic calponin (green). The scale bar = 50 µm. In b, CL14 cells double-immunostained for vWF
(left) and basic calponin (middle), along with
the merged image (right). Some cells are positive only for
vWF (arrowhead), some are positive only for basic calponin
(open arrow), and some are positive for both markers
(closed arrows). The scale bar = 20 µm. In
c, cloned HUVE-DCs were cultured for the indicated periods
in the absence of FGF-1 and heparin, and expression of basic calponin
and vWF mRNA was examined by semiquantitative RT-PCR/Southern blot
analyses (top). The relative expression level of calponin
mRNA was plotted after normalization with GAPDH expression
(bottom); the maximum level detected was defined as 100%
expression. In d, CL14 cells were cultured for 4 days in the
absence of FGF-1 and heparin and then for another 4 days (until day 8)
in the same medium without ( ) or with (+) FGF-1 and heparin.
Expression of basic calponin and SM22
mRNA was then assessed by
semiquantitative RT-PCR/Southern blot analyses as in panel c
(inset).
, followed a similar time course (data not shown). These effects of FGF deprivation proved to be reversible. In the experiment shown in Fig. 2d, FGF-1 deprivation increased expression of
basic calponin and SM22
mRNA by day 4. However, addition of
FGF-1 at that time reversed the increase, almost eliminating expression of basic calponin and SM22
mRNA by day 8. A similar reversal of
SMC-like-differentiation by FGF was also observed in parental HUVE-DCs
(data not shown). In addition, as in the parental HUVE-DC cultures
(Fig. 1c), expression of vWF mRNA increased in both
FGF-deprived CL1 and CL14 cells (Fig. 2c).
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Fig. 3.
ET-1-evoked contraction of HUVE-DC-derived
SMC-like cells. In a, HUVE-DCs were cultured in the
absence of FGF-1 and heparin and for the indicated periods, after which
their expression of ET-receptor type A and ET-1 mRNA was examined
by semiquantitative RT-PCR/Southern blot analyses (left
panels). The relative levels of each mRNA were plotted after
normalization with GAPDH expression (right panels). For ET-R
type A, the maximum level was defined as 100% expression; for ET-1,
the initial level was defined as 100% expression. In b,
after depriving cells cultured on collagen-coated plastic plates of
FGF-1 and heparin for 3 weeks, they were plated on gels formed of type
I collagen and incubated with (middle and right)
or without (left) ET-1 (250 nM) for 30 min. The
cells in the right panel were exposed to ET-R inhibitors (30 µM of BQ123 and 10 µM of BQ788) for 10 min
prior to application of ET-1. Phase-contrast micrographs are shown. The
scale bar = 50 µm. In c, HUVE-DCs cultured
in growth medium containing FGF-1 (F) or in FGF-free medium
for 3 weeks (D) were harvested and embedded into gels formed
of type I collagen. Their capacity to contract was analyzed by
comparing the relative volumes of gels in which the cells were (+) or
were not ( ) treated with ET-1 (250 nM) for 3.5 h. *,
p < 0.05; Student's t test. In
d, HUVE-DCs cultured in FGF-free medium for 3 weeks were
harvested, embedded into collagen gel, and analyzed for their capacity
to contract as in panel c. ET-R inhibitors (30 µM of BQ123 and 10 µM of BQ788) were added
to some cultures for 30 min prior to application of ET-1. *,
p < 0.05; Student's t test. In
e, HUVE-DCs and CL14 cells cultured in FGF-free medium for 3 weeks, or HPASMCs cultured in FGF-free medium, were plated on type I
collagen gels. The cells were treated (+) or not treated (
) with
ET-1, after which cell contraction was analyzed by measuring the size
of the cell areas on the gels. *, p < 0.05; Student's
t test.
) with ET-1 were
compared. We found that the area occupied by HPASMCs decreased to
50.4% of control upon exposure to ET-1, whereas the areas occupied by the differentiated HUVE-DCs and CL14 cells declined to 73.1 and 74.9%
of control, respectively (Fig. 3e).
family. Because both migrating
and resting ECs reportedly express activin A, which inhibits
endothelial cell proliferation (34), we examined expression of activin
A and other genes related to activin A signaling (Fig.
4a). We determined that
activin A expression in CL14 cells increased after FGF-1 deprivation
and that reapplying FGF-1 to the cells reversed this up-regulation
(Fig. 4a). The same results were obtained with
parental cells (data not shown). Expression of mRNAs encoding
activin-inducible inhibitors of activin signaling, i.e.
follistatin (35-37) and Smad7 (18, 38-42), was also reversibly
increased after FGF-1 deprivation (Fig. 4a), indicating that
endogenous activin A signaling is activated in FGF-deprived cells.
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Fig. 4.
Importance of activin A signaling in the
differentiation of HUVE-DCs into SMC-like cells. In a,
CL14 cells were cultured for 4 days in the absence of FGF-1 and heparin
and then for another 4 days (until day 8) in the same medium without
( ) or with (+) FGF-1 and heparin. Expression of activin A mRNA
and its related genes was examined by semiquantitative RT-PCR/Southern
blot analyses (left panels). Relative expression levels were
plotted after normalization with GAPDH expression; the initial level
was defined as 100% expression (right panel). In
b, HUVE-DCs were cultured for 5 days with or without FGF-1.
Follistatin (1 µg/ml) was added to half of the cultures without FGF-1
once every 12 h throughout the culture period. The expression of
mRNAs encoding SMC and EC markers was examined by semiquantitative
RT-PCR/Southern blot analyses. In c, HUVE-DCs were cultured
for 7 days with or without FGF-1, with (+) or without (
) activin A
(100 ng/ml). Expression of marker genes was examined as in panel b. In d, HUVE-DCs were cultured for 9 days without FGF-1 in the presence (left) or absence
(right) of activin A (100 ng/ml). The cells were then
double-immunostained for vWF (red) and basic calponin
(green). The scale bar = 50 µm. In
e, CL1 and CL14 cells were cultured for 4 days in the
absence of FGF-1 and heparin and then for another 4 days (until day 8)
in the same medium with (+) or without (
) FGF-1 and heparin.
Expression of activin A mRNA was examined by semiquantitative
RT-PCR/Southern blot analyses (left panels). Relative
expression levels were plotted after normalization with GAPDH
expression (right panel). In f, CL1 and CL14
cells were cultured for 7 days with or without FGF-1 in the presence or
absence of activin A (100 ng/ml), after which marker gene expression
was examined as in panel b.
expression (Fig.
4b), although it had no effect on vWF expression (Fig.
4b). This result strongly suggested that endogenous activin
A signaling is responsible for inducing the SMC-like phenotype in
FGF-deprived HUVE-DCs. Consistent with that interpretation, addition of
exogenous activin A to FGF-deprived cultures induced expression of
basic calponin and SM22
mRNA (Fig. 4c), as well as
calponin protein (Fig. 4d). Interestingly, when we
simultaneously added FGF-1, the activity of exogenous activin A was
suppressed (Fig. 4c).
mRNA in both CL1 and CL14 cells but to a much greater extent in the latter (Fig. 4f). Thus, activin A signaling appears to
correlate with SMC-like differentiation of HUVE-DCs at the cell
population level, suggesting that such differentiation is determined,
at least in part, by the level of activin A expression following FGF deprivation.
mRNAs (Fig. 5d) and of basic calponin protein (green) (Fig. 5e). There was no difference in the
level of vWF mRNA expression in control and Smad7-overexpressing
cells.
View larger version (55K):
[in a new window]
Fig. 5.
Inhibition of HUVE-DC differentiation into
SMC-like cells by overexpression of Smad7. a,
expression of FLAG-Smad7 in HUVE-DCs infected with various amounts of
AdCMV-FLAG-Smad7. The cells were then lysed, and expression
of the target protein was analyzed by immunoblotting.
AdCMV-LacZ was used in control experiments. b,
activation of Smad2 by activin A. HUVE-DCs were treated with activin A
(100 ng/ml) for the indicated times, after which phosphorylated
(upper) and total (lower) Smad2 were detected by
immunoblotting. In c, HUVE-DCs were transfected with
AdCMV-Smad7 (panels 1 and 2) or
AdCMV-LacZ (panels 3 and 4), after
which they were left untreated (panels 1 and 3)
or exposed to activin A (100 ng/ml) for 1 h (panels 2 and 4). The cells were then fixed, and subcellular
localization of Smad2 was detected using anti-Smad2 monoclonal antibody
and a fluorescently labeled secondary antibody. The scale
bar = 10 µm. d, inhibition of SMC marker
expression by overexpression of Smad7. HUVE-DCs were infected with
AdCMV-Smad7 or AdCMV-LacZ and then cultured for 5 days in FGF-1-free medium containing activin A (100 ng/ml). Expression
of mRNAs encoding SMC and EC markers was examined by
semiquantitative RT-PCR/Southern blot analyses. In e,
inhibition of SMC differentiation by overexpression of Smad7. HUVE-DCs
were infected with AdCMV-Smad7 (panels 1 and
3) or AdCMV-LacZ (panels 2 and
4) and cultured in FGF-1-free medium containing activin A
(100 ng/ml) for 7 days. Phase-contrast (1 and 2)
and fluorescent (3 and 4) micrographs of the
cells double-immunostained for anti-vWF (red) and basic
calponin (green) are shown. The scale bar = 50 µm.
family members, i.e. TGF-
1
and TGF-
3, have been shown to induce expression of SMC markers in bovine aortic ECs (8) and mesenchymal cells (10), we examined the
effects of neutralizing anti-TGF-
1, -
2,and -
3 antibody on the
expression of SMC marker genes. We found that the antibody did not
significantly affect expression of these genes, nor did direct addition
of TGF-
1 (16 ng/ml) or BMP-4 (50 ng/ml) to the cultures (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, although the increases were more modest than were seen in
HUVE-DCs (data not shown). These results strongly suggest that ECs
derived from adult human blood vessels also retain the potential to
differentiate into SMC-like cells. To clarify this issue, however, more
detailed investigation, including characterization of cell
differentiation at the single cell level, will be essential.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Makiko Fujii at the Department of Biochemistry, The Center Institute of the Japanese Foundation for Cancer Research, Tokyo, Japan and Dr. Atsuhito Nakao at the Allergy Research Center, Juntendo University School of Medicine, Tokyo, Japan for AdCMV-Smad7, and we thank Dr. Peter ten Dijke at the Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, Netherlands for anti-phosphorylated smad2 antibody.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Age Dimension
Research Center, National Institute of Advanced Industrial Science and
Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Tel.: 81-298-61-6504; Fax: 81-298-61-6149;
E-mail: imamura-toru@aist.go.jp.
Published, JBC Papers in Press, November 1, 2002, DOI 10.1074/jbc.M207329200
2 A. Ishisaki and T. Imamura, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: EC, endothelial cell; SMC, smooth muscle cell; FGF, fibroblast growth factor; TGF, transforming growth factor; PDGF, platelet-derived growth factor; HUVE-DC, human umbilical vein endothelium-derived cells; HUVEC, human umbilical vein ECs; HPAECs, human adult pulmonary artery endothelial cells; HPASMCs, human adult pulmonary artery smooth muscle cells; BMP, bone morphogenetic protein; ET, endothelin; ET-R, endothelin receptor; vWF, von Willebrand factor; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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