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Cell Biology Laboratory, Department of Gynecology and Obstetrics, University of Göttingen Medical School, 37075 Göttingen; and
* Institute of Molecular Medicine, Tumor Biology Center, 79106 Freiburg, Germany
1Correspondence: Institute of Molecular Medicine, Tumor Biology Center, D-79106 Freiburg, Germany. E-mail: augustin{at}angiogenese.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: EC • SMC • spheroid • vascular endothelial growth factor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Correspondingly, exogenous PDGF-B has been shown to
promote pericyte recruitment to the immature retina vasculature of
newborn rats (2)
. These experiments also demonstrated that
mural cells stabilize the immature neovasculature, rendering
endothelial cells (EC) independent of the activities of survival
factors such as vascular endothelial growth factor (VEGF) and limiting
the plasticity window that allows remodeling and pruning of a newly
formed vascular bed (2
, 3)
. Another receptor/ligand system
involved in vessel assembly and maturation is the Tie-2/angiopoietin
system (4
, 5)
. The ligand for the receptor tyrosine kinase
Tie-2 angiopoietin 1 (Ang-1) has been identified as an endothelial cell
specific growth factor that is proangiogenic, acts as an endothelial
cell survival factor, and promotes stabilization of blood vessels
through endothelial cell contacts with mural cells (6
7
8)
.
Ang-1 reduces vascular permeability even in the presence of the
proangiogenic and permeability-promoting cytokine VEGF
(9)
. Ang-2 has been characterized as a competitive
functional antagonist of Ang-2 that binds Tie-2 without transducing an
activating signal (10)
. As a consequence, Ang-2 is
believed to act as a vessel-destabilizing agent that either induces
vessel regression (in the absence of proangiogenic activity) or
facilitates angiogenesis (in the presence of proangiogenic activity)
(4
, 5)
. Most recent findings on the functions of molecules that play critical roles in the process of vascular morphogenesis have been generated through genetic manipulation experiments in mice by either specifically deleting the function of a candidate gene (loss of function) or by transgenically overexpressing a gene of interest (gain of function). As sophisticated as these experimental strategies are, the early embryonic lethality of mice with targeted mutations of genes that are critical for vascular morphogenetic events greatly reduces examination of the resulting phenotypes to morphological analyses and limits detailed mechanistic experiments.
Mechanistic experiments on specific endothelial cell functions have
been performed in great detail using monolayer cell culture techniques
on plastic substrata or components of the extracellular matrix. These
studies have contributed to understanding the complexity of the
vascular endothelium. Great progress has been made in identifying
molecular determinants of activated endothelial cells as they are
expressed during inflammation, atherosclerosis, or angiogenesis
(11
12
13)
. However, the reductionist approach of standard
monolayer cell culture strategies and the inherent dedifferentiation of
primary cultures of endothelial cells have largely precluded in
vitro studies of complex endothelial cell functions as they are
associated with vessel assembly, maturation, and organotypic as well as
caliber-specific differentiation. We have recently developed a
3-dimensional spheroidal system of endothelial cell differentiation
(14)
and in vitro angiogenesis
(15)
. Based on the unique properties of this model, we
hypothesized it might be possible to further develop this cell culture
system toward a proper in vitro representation of the
3-dimensional assembly of a normal blood vessel. Consequently, we
devised experiments aimed at establishing organized coculture spheroids
of endothelial cells and smooth muscle cells (SMC) with a luminal
aspect, a polarized endothelial cell monolayer, and an underlying
multilayered assembly of smooth muscle cells. After developing a model
that fulfills these requirements, we pursued experiments to study
paracrine interactions between endothelial cells and smooth muscle
cells that control the quiescent phenotype of endothelial cell and
regulate the functions of angiogenic cytokines.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
Endothelial cell growth medium (ECGM), endothelial cell growth
supplement (human umbilical vein endothelial cell culture), and human
smooth muscle cell growth medium (HSMCGM) were purchased from Promocell
(Heidelberg, Germany). Fetal calf serum (FCS) was obtained from
Biochrom (Berlin, Germany). Human umbilical vein endothelial cells
(HUVEC) were freshly isolated from human umbilical veins of newborn
babies by collagenase digestion. Cells were cultured at 37°C in 100
mm tissue culture dishes in ECGM containing 10% heat-inactivated fetal
calf serum and frozen in liquid nitrogen at passage 2 or 3. Only HUVE
cells cultured from passage 4 to 8 were used for experiments. Human
umbilical artery smooth muscle cells were purchased from Promocell and
cultured in HSMCGM at 37°C in 100 mm tissue culture dishes up to
passage 9.
Generation of endothelial cell, smooth muscle cell, and coculture
spheroids
Endothelial cell and smooth muscle cell spheroids of defined
cell number were generated as described previously (14)
.
In brief, SM or HUVE cells were suspended in corresponding culture
medium containing 0.25% (w/v) carboxymethylcellulose and seeded in
nonadherent round-bottom 96-well plates (Greiner, Frickenhausen,
Germany). Under these conditions, all suspended cells contribute to the
formation of a single spheroid per well of defined size and cell number
(standard size: 2250 cells/spheroid; in vitro angiogenesis:
750-1000 cells/spheroid). To generate coculture spheroids, equal
amounts of suspended SM and HUVE cells (standard size: 1125 SMC and
1125 HUVEC per spheroid; in vitro angiogenesis: 500 SMC and
500 HUVEC per spheroid) were mixed and seeded in nonadherent
round-bottom 96-well plates as described above. Spheroids were cultured
for at least 24 h and used for the corresponding experiments.
In vitro angiogenesis assay
In vitro angiogenesis in collagen gels was
quantitated using endothelial cell, smooth muscle cell, and coculture
spheroids as described previously (15)
. In brief,
spheroids containing 750-1000 cells were generated overnight, after
which they were embedded into collagen gels. A collagen stock solution
was prepared prior to use by mixing 8 vol acidic collagen extract of
rat tails (equilibrated to 2 mg/ml, 4°C) with 1 vol 10x EBSS (Gibco
BRL, Eggenstein, Germany); 
1 vol 0.1 N NaOH to adjust the pH to
7.4. This stock solution (0.5 ml) was mixed with 0.5 ml room
temperature medium (ECGM basal medium [PromoCell] with 40% FCS
[Biochrom, Berlin, Germany]) containing 0.5% (w/v)
carboxymethylcellulose to prevent sedimentation of spheroids prior to
polymerization of the collagen gel, 50 spheroids, and the corresponding
test substance. The spheroid containing gel was rapidly transferred
into prewarmed 24-well plates and allowed to polymerize (1 min), after
which 0.1 ml ECGM basal medium was pipetted on top of the gel. The gels
were incubated at 37°C, 5% CO2, and 100%
humidity. After 24 h, in vitro angiogenesis was
digitally quantitated by measuring the length of the sprouts that had
grown out of each spheroid (ocular grid at 100x magnification) using
the digital imaging software DP-Soft (Olympus, Germany) analyzing at
least 10 spheroids per experimental group and experiment.
Fluorescent cell labeling
SMC and HUVEC were labeled using the fluorescent dyes PKH26 (red
fluorescence) and PKH67 (green fluorescence) following manufacturer’s
instructions. After trypsinization, suspended cells were washed once
with HBSS, membrane labeled with PKH26 or PKH67 for 5 min, and washed
three times using corresponding culture medium. Quality of cell
labeling was examined using fluorescence microscopy.
Ultrastructural analysis
Spheroids were fixed in Karnovsky’s fixative, postfixed in
1.0% osmium tetroxide, dehydrated in a graded series of ethanol, and
embedded in Epon. Sections of 0.5 µm were cut and stained with azure
11 methylene blue for light microscopic evaluation. Ultrathin sections
(50–80 nm) were cut, collected on copper grids, and automatically
stained with uranyl acetate and lead citrate for observation with a
Zeiss EM 10 electron microscope.
For quantitation of interendothelial junctional complexes in surface spheroid endothelial cells, all junctional complexes of 20 randomly selected spheroids per experimental group in two independent preparations were counted. Results were expressed as the number of junctional complexes per 100 surface monolayer endothelial cells (analysis of at least 200 EC per experimental group).
Morphological and immunohistochemical analysis
Spheroids were harvested and centrifuged for 3 min at 200
g. Cultured monolayer cells were harvested by trypsinization
and collected by centrifugation. Spheroids and pelleted monolayer cells
were fixed in HBSS containing 4% paraformaldehyde and processed for
paraffin embedding; after dehydration (graded series of ethanol and
isopropanol, 1 h each), the specimens were first immersed with
paraffin I (melting temperature 42°C) for 12 h at 60°C.
Spheroids and monolayer cells were again collected by centrifugation
and immersed with paraffin II (melting temperature 56°C) for 12 h at 70°C. Finally, the resulting paraffin block was cooled to room
temperature and trimmed for sectioning. For histochemical analyses,
paraffin sections (4 µm) were cut, deparaffinized, and rehydrated.
Sections were then incubated with 3%
H2O2 in
H2O to inhibit endogenous peroxidase. After
washings in phosphate-buffered saline, the sections were incubated for
30 min with blocking solution (10% normal goat serum), followed by
incubation with the corresponding primary antibody in a humid chamber
at 4°C overnight. Then they were incubated with secondary antibody
(biotinylated goat anti-rabbit immunoglobulin or biotinylated goat
anti-mouse immunoglobulin antibody; Zymed, San Francisco, Calif.),
exposed to streptavidin peroxidase, developed with diaminobenzidine as
substrate, and weakly counterstained with hematoxylin.
Detection of apoptotic cells in spheroids
Apoptotic cells were visualized by histochemical detection of
nucleosomal fragmentation products (TUNEL) applying the In Situ Cell
Death Detection Kit (Boehringer Mannheim, Germany) following the
manufacturer’s instructions. In brief, nucleosomal fragmentation
products in sections of paraffin-embedded spheroids were detected after
deparaffination and proteinase K digestion by 3' end labeling with
fluorescein-dUTP using terminal deoxynucleotidyl transferase. Labeling
was visualized either directly by fluorescence microscopy or indirectly
after incubating the sections with peroxidase-labeled anti-fluorescein
antibody and developing with diaminobenzidine as substrate.
DNA fragmentation enzyme-linked immunoassay (ELISA)
Quantitation of fragmented DNA was performed by ELISA (Cell
Death Detection ELISA Kit; Boehringer Mannheim, Germany). Fragmented
DNA of 10 spheroids was extracted by lysis for 60 min at room
temperature with vigorous shaking. The extracts were centrifuged for 10
min at 13,000 g and 300 µl of the supernatant was
incubated with peroxidase-labeled anti-DNA antibody and biotinylated
anti-histone antibody in streptavidin-coated microtiter plates
following the manufacturer’s instructions. After washing, binding of
mono- and oligonucleosomal DNA was visualized by developing with the
peroxidase substrate ABTS
(2,2'-azino-di[3-ethylbenzthiazolin-sulfonate]). Plates were analyzed
at 405 nm using an automated microtiter plate reader (EAR 400AT, SLT
Lab instruments, Austria).
Statistical analysis
All results are expressed as mean ± SD.
Differences between experimental groups were analyzed by unpaired
Student’s t test. P values <0.05 were
considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, we developed a spheroidal
coculture system of endothelial cells and smooth muscle cells aiming at
mimicking the correct 3-dimensional assembly of the normal vessel wall.
Equal numbers of human umbilical vein EC and human umbilical artery SMC
were mixed and suspended in nonadhesive 96-well round-bottom plates in
methocel containing medium. Essentially all of the cells in one well
sediment and contribute to the formation of a single cellular
aggregate, which was found to spontaneously organize into a core of SMC
and a surface layer of EC. Differentiation of the coculture spheroids
was traced by staining endothelial cells for CD31 expression
(Fig. 1
). Complementary results were obtained by staining SMC for the
expression of 
-smooth muscle actin. Solo EC spheroids stained
uniformly for CD31 with the surface EC stained more intense than the
center EC (Fig. 1A
). In contrast, solo SMC spheroids did not
stain for CD31 (Fig. 1B
). Coculture spheroids of EC and SMC
differentiate within 2 days to establish a core of SMC and a surface
layer of EC (Fig. 1C
). The surface layer differentiates over
time to form a flat monolayer of cells within 4 days (Fig. 1D
). Thus, coculture spheroids of EC and SMC differentiate
in a way that resembles the 3-dimensional organization of the normal
vessel wall in an inside-out orientation of luminal side (medium),
endothelial cell lining, and multilayered SMC core. This compartmental
organization was found to be calcium dependent. When coculture
spheroids were allowed to establish for 24 h and then treated with
EGTA for another 24 h, spheroidal organization was found to be
completely disrupted, with EC and SMC evenly distributed within the
spheroid (Fig. 1E
). Likewise, when different endothelial
cell populations were mixed (HUVEC and murine endothelioma cells BEND),
the cells did not mix and formed clustered aggregates of individual
cell populations, demonstrating the specificity of the morphogenetic
interactions that occur in EC/SMC coculture spheroids (Fig. 1F
).
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Formation of interendothelial junctional complexes is enhanced in
EC/SMC coculture spheroids
Based on the observed spontaneous organization of coculture
spheroids of EC and SMC, we next asked whether SMC in coculture
spheroids control phenotypic properties of the surface endothelial cell
layer. Sections of EC spheroids and EC/SMC coculture spheroids cultured
for 1 and 4 days were examined ultrastructurally and the number of
interendothelial electron dense junctional complexes was quantitated
(Fig. 2
). The number of interendothelial cell junctional complexes of the
surface endothelial cell monolayer increased over time in both solo EC
spheroids and EC/SMC coculture spheroids. Endothelial cells in
coculture spheroids, however, established more than twice as many
junctional complexes after 4 days as solo EC spheroids
(P<0.05; Fig. 2A
).
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Endothelial PDGF-B expression is completely down-regulated in
EC/SMC coculture spheroids over time
EC cultured as monolayers express abundant levels of PDGF-B under
various culture conditions, which has repeatedly been interpreted to
reflect the inability to properly mimic the in vivo
quiescent EC phenotype in tissue culture (16
, 17)
.
Corresponding in vivo experiments have demonstrated that
endothelial cell PDGF-B expression is restricted to immature capillary
endothelial cells and to the endothelium of growing arteries
(18)
. We consequently analyzed PDGF-B expression in
monolayer EC and in spheroids of EC and SMC, applying histochemical
techniques in order to identify the PDGF-B expressing cells. Sections
of embedded confluent and subconfluent monolayers of EC expressed very
high levels of PDGF-B (Fig. 3A
, B
). Similarly, EC cultured in solo EC spheroids diffusely
express abundant amounts of PDGF-B after 1 or 4 days in culture (Fig. 3C
, D
). SMC in spheroids do not express PDGF-B (Fig. 3E
, F
). Coculture spheroids of EC and SMC have detectable
levels of PDGF-B in their center as well as in the surface monolayer
after 1 day in culture (Fig. 3G
). No PDGF-B expression is
detectable in EC/SMC coculture spheroids after 4 days. The surface
monolayer of EC, which is in contact with the underlying EC, becomes
completely PDGF-B negative in these coculture spheroids (Fig. 3H
). Histochemical analysis of PDGF-B expression in solo and
coculture spheroids was complemented by ELISA quantitation of PDGF-B
protein in the supernatants of 4 day solo and coculture spheroids.
Significant levels of PDGF-B protein were detected in the supernatant
of solo EC spheroids. In contrast, PDGF-B protein concentrations were
beyond detection level in the supernatants of SMC spheroids as well as
EC/SMC-coculture spheroids (data not shown), confirming the
histochemically determined down-regulation of EC PDGF-B synthesis in
coculture with SMC.
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Coculture of EC and SMC in spheroids inhibits EC apoptosis induced
by serum starvation
Contact of endothelial cells with mural cells (pericytes, smooth
muscle cells) has been shown to limit a plasticity window for vessel
remodeling and to render endothelial cells independent of the
activities of survival factors such as VEGF, FGF-2, or Ang-1 (2
, 19)
. We consequently analyzed whether EC apoptosis is affected
by the presence of SMC in EC/SMC coculture spheroids. Solo EC and SMC
spheroids as well as EC/SMC coculture spheroids were cultured for 2
days under low serum conditions. Under these conditions, EC in solo EC
spheroids undergo massive apoptosis, as evidenced by DNA fragmentation
ELISA measuring the amount of fragmented DNA of 10 individual spheroids
(EC control set as 100%; Fig. 4A
, B
). Treatment of EC spheroids with EGTA (disruption of
Ca-dependent cell–cell contacts) or anti-FGF-2 antibody (inhibition of
endogenous FGF-2) increased the level of EC beyond the linear scale of
the apoptosis ELISA (comparison of ELISA and TUNEL; Fig. 4A
, B
, E
). Likewise, treatment of EC spheroids with FGF-2 decreased the
level of apoptosis significantly by 50% (P<0.005, Fig. 4A
). In contrast, SMC cultured in spheroids have very low
baseline levels of apoptosis that are barely influenced by exposure to
EGTA, FGF-2, or -FGF-2 (Fig. 4A
, C
, F
). Cocultures of
equal numbers of EC and SMC had significantly lower levels of apoptosis
as the calculated mean of solo EC and SMC spheroids (EC: 100%, SMC:
8%, EC/SMC calculated: 54%; EC/SMC observed: 18%; Fig. 3A
, D
). Treatment of coculture spheroids with EGTA increased the
observed level of apoptosis toward the calculated level (Fig. 3A
, G
). Together, these findings strongly suggest that the presence of
SMC in the coculture spheroids stabilizes EC to reduce the levels of EC
apoptosis, although it must be realized that EC have very different
growth configurations in solo and coculture spheroids, which may also
affect EC apoptosis.
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EC/SMC contacts in coculture spheroids inhibit VEGF-induced
endothelial CD34 expression
Most EC in vivo express the cell surface
glycoprotein CD34 (20
, 21)
. Upon transfer of EC in tissue
culture, however, CD34 is rapidly down-regulated (22)
. We
have recently shown that VEGF selectively stimulates the surface EC in
3D spheroids to reexpress CD34 (14)
. Based on these
findings, we analyzed endothelial CD34 expression in EC and EC/SMC
spheroids as a functional readout for VEGF-dependent activation of EC.
Untreated EC cultured in spheroids do not express CD34 (Fig. 5A
, B
). Treatment of EC spheroids with VEGF induces the
surface EC to express CD34 (Fig. 5B
). This effect can be
quantitated by counting the number of positive cells of the surface
monolayer (Fig. 5A
). EC cocultured with SMC in spheroids do
not express CD34 (Fig. 5A
, C
) and cannot be induced to
express CD34 by stimulation with VEGF (Fig. 5A
, E
),
suggesting that the surface layer of EC has become refractory to the
stimulation with VEGF upon contact with SMC. To analyze whether
cell–cell contacts between EC and SMC are involved in regulating EC
responsiveness toward VEGF in the presence of SMC, we reduced the
number of cellular contacts between EC and SMC by coculturing four
times as many EC as SMC in coculture spheroids, which leads to the
formation of a multilayered surface of EC. Stimulation of these 4:1
EC/SMC coculture spheroids with VEGF significantly induced surface EC
to express CD34 compared to VEGF-treated 1:1 EC/SMC coculture spheroids
(P<0.001; Fig. 5A
).
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VEGF stimulation fails to induce sprouting of EC originating from
collagen-embedded EC/SMC coculture spheroids
To further analyze the effects of SMC on EC effector functions and
VEGF responsiveness, we performed experiments in gel angiogenesis with
EC and EC/SMC spheroids. Spheroids were embedded in collagen gels and
stimulated with VEGF. The cumulative length of outgrowing
capillary-like sprouts was quantitated after 24 h (for unambiguous
identification of cells, EC and SMC were labeled with different
fluorescent dyes prior to the formation of coculture spheroids; see
Materials and Methods).
VEGF acts as a potent inducer of sprouting angiogenesis originating
from gel-embedded EC spheroids (fourfold higher cumulative sprout
length; P<0.001; Fig. 6A
, B
, F
). In contrast, VEGF stimulation of SMC embedded as
spheroids did not induce sprouting of SMC into the collagen gel within
24 h (Fig. 6A
, C
, G
). There was no sprouting of cells
from EC/SMC coculture spheroids (Fig. 6A
, D
). Corresponding
to the nonresponsiveness of EC in EC/SMC coculture spheroids in the
CD34 induction experiments, VEGF had no effect on sprouting of EC into
the collagen originating from EC/SMC coculture experiments (Fig. 6A
, H
). However, when changing the ratio of EC to SMC to
4:1, we observed a significant induction of EC sprouting angiogenesis
by VEGF originating from EC/SMC coculture spheroids (compared to 1:1
EC/SMC spheroids; P<0.001; Fig. 6A
, E
, I
).
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Costimulation of collagen gel-embedded EC/SMC spheroids with Ang-2
and VEGF induces EC sprouting
Based on the observed nonresponsiveness of EC toward VEGF in
EC/SMC coculture spheroids, we set out experiments aimed at restoring
VEGF responsiveness of EC cocultured in the presence of SMC. These
experiments led to costimulation in vitro angiogenesis
experiments with VEGF and Ang-2. Ang-2 has been identified as a
vessel-destabilizing cytokine that acts by functionally antagonizing
Ang-1-mediated vessel maturation (5
, 10)
. When applied
individually, neither VEGF nor Ang-2 was able to induce EC sprouting
originating from EC/SMC coculture spheroids (Fig. 7A
, B
, C
, D
, G
, H
, I
). However, costimulation of EC/SMC
coculture spheroids with VEGF and Ang-2 induced EC sprouting (Fig. 7A
, E
, J
). PMA served as a positive control in these
experiments, stimulating both EC as well as SMC outgrowth by directly
stimulating protein kinase C (Fig. 7F
vs. Fig. 7K
).
|
In line with the presumed function of Ang-2 as an Ang-1-antagonizing,
vessel-destabilizing agent (5)
, the observed synergistic
effect of Ang-2 and VEGF on in vitro angiogenesis suggested
that Ang-2 might act as a facilitator of VEGF function. We consequently
analyzed the expression status of endogenous Ang-1 and Ang-2 in solo EC
and SMC spheroids as well as in coculture spheroids. To assess relative
ratios of Ang-1 and Ang-2, we used a quantitative coamplifying ratio
reverse transcriptase-polymerase chain reaction (RT-PCR; ref
23
). EC and SMC were found to express a complementary,
non-overlapping pattern of angiopoietin production. EC in spheroids
express Ang-2, whereas SMC in spheroids express Ang-1 (Fig. 7L
). Coculture spheroids (1:1) were found to express
approximately equimolar ratios of Ang-1 and Ang-2 (densitometric
Ang-2/Ang-1 ratio: 0.87±0.15; n=4). Stimulation of
coculture spheroids with FGF-2 led to a significant shift of the
Ang-2/Ang-1 ratio toward Ang-2 (2.36±0.19; P<0.001;
n=4), corresponding to previously reported findings on the
induction of Ang-2 by FGF-2 (26)
. In contrast, addition of
VEGF did not change the relative ratio of Ang-2 to Ang-1 mRNA
(0.98±0.08; n=4), confirming the nonresponsiveness of
EC/SMC coculture spheroids to VEGF stimulation.
Ang-2 stimulates endothelial cells in the absence of smooth muscle
cells
Based on the observed synergism of Ang-2 and VEGF in mediating EC
sprouting in the presence of Ang-1 expressing SMC, we next performed
experiments to assess possible direct endotheliotropic functions of
Ang-2 in the absence of SMC. We performed EC lateral sheet migration
experiments (Fig. 8A
) and gel angiogenesis experiments using solo EC spheroids
(Fig. 8B
). Surprisingly, both experimental approaches
demonstrated that Ang-2 can directly stimulate EC in the absence of
SMC, capable of dose-dependently stimulating EC migration and sprouting
angiogenesis (Fig. 8)
. Addition of sTie-2 completely blocked
Ang-2-induced sprouting angiogenesis, confirming the specificity of
Ang-2 induced endothelial cell activation.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Numerous coculture systems of EC and SMC have been developed to study
paracrine interactions in the vessel wall. These include planar
coculture models of cells cultured together in the same dish, bilayer
coculture, two-compartment filter systems, and agarose cocultures
(16
, 17
, 25
26
27)
. These studies have shown that EC and SMC
regulate each other’s quiescent phenotype. EC-derived PDGF-BB controls
mural cell recruitment and differentiation (1
, 26)
. In
turn, mural cell-derived, activated transforming growth factor ß
(TGF-ß) contributes to the maintenance of the quiescent EC phenotype
(28
, 29)
.
Advancing a spheroidal EC cell culture system developed in our
laboratory (14)
, we have established in the present study
a spheroidal coculture system of EC and SMC that is capable of
mimicking the 3-dimensional assembly of a blood vessel with a luminal
aspect, a polarized endothelial cell monolayer, and an underlying
multilayered assembly of smooth muscle cells. These coculture spheroids
can be regarded as an inside-out assembly of a resting vessel wall in
which SMC control the quiescent phenotype of the EC monolayer.
Organized EC/SMC coculture spheroids form within 2 days by simply
pipetting them together in 96-well round-bottom dishes under
nonadhesive conditions. The spontaneous differentiation of the
coculture spheroids thus suggests a distinct morphogenetic interaction
between EC and SMC to organize in a vessel wall like structure.
In
analyzing the properties of the cells in the coculture spheroids and the
interactions between the different cell types, we focused on
SMC-mediated effects on the surface monolayer of EC. Several lines of
experimental evidence demonstrated that SMC control the quiescent
phenotype of EC through direct cell–cell contacts: 1) EC
coculture in contact with SMC establish an increased number of
interendothelial junctional complexes, 2) EC cocultured in
spheroids with SMC down-regulate their expression of PDGF-B (16
, 17)
, and 3) apoptosis of EC is reduced in coculture
spheroids in the presence of SMC. Together, these findings suggest that
the 3-dimensional assembly of EC and SMC mimics many of the
physiological EC properties as they are expressed by the quiescent
organ vasculature.
Given recent advances in the molecular mechanisms that control vessel
assembly and maturation during vascular morphogenetic events (30
, 31)
, we applied the EC/SMC coculture model to study effects of
vascular morphogenetic cytokines on EC functional properties cocultured
with SMC. We found that coculture of EC with SMC completely abrogated
VEGF responsiveness, which was demonstrated by a lack of endothelial
CD34 inducibility in the presence of SMC as well as an inhibition of
sprouting angiogenesis in response to VEGF. The mechanism of
SMC-mediated nonresponsiveness to VEGF is being investigated in our
laboratory. Activation of latent TGF-ß has been shown to mediate some
of the quiescence effects exerted by SMC on EC (28
, 29)
.
However, exogenous addition of TGF-ß resulted in partial inhibition
of EC activation, but in none of our experiments was able to mediate
nonresponsiveness to VEGF to an extent as direct coculture with SMC
(data not shown). Recent experiments suggest that endothelial
cell-derived PlGF is required for VEGF responses in the adult
(32)
. PlGF deficient mice have no apparent developmental
or reproductive phenotype, but are not able to properly respond to VEGF
(32)
. The mechanism of this interaction has not been
uncovered, but the interaction of PlGF and VEGF in mediating VEGF
responsiveness suggests that VEGF responsiveness is probably not
limited primarily by presentation of VEGF receptors.
Another cytokine that may be able to mediate VEGF responses is Ang-2.
Ang-2 has been identified as a functional antagonist of the Tie-2
ligand Ang-1 (10)
. Presumably, Ang-2 destabilizes
interactions of EC with mural cells by competitively binding Tie-2
without transducing an activating signal. Correspondingly, Ang-2
functions are considered context dependent, either facilitating
angiogenesis (in the presence of angiogenic activity) or inducing
vessel regression (in the absence of angiogenic activity) (4
, 5)
. Ang-2 is produced primarily by endothelial cells and thus
seems to act as an autocrine regulator of vessel destabilization
(24
, 33)
. In line with the antagonistic model of Ang-2
function, costimulation of EC/SMC coculture spheroids with VEGF and
Ang-2 led to the induction of sprouting angiogenesis in collagen gels,
whereas neither VEGF nor Ang-2 was able to induce sprouting
angiogenesis on its own. These findings demonstrate for the first time
an in vitro function of Ang-2 on EC and can be interpreted
as reflecting a facilitating role of Ang-2 for VEGF responsiveness in
the presence of Ang-1-expressing SMC. When analyzing the effects of
Ang-2 on solo EC populations, however, we made the puzzling observation
that Ang-2 stimulates lateral sheet migration of EC as well as
sprouting angiogenesis of gel-embedded EC spheroids. Collectively,
these findings support a hypothetical model whereby Ang-2 function is
context dependent in a way that it may act as an antagonistic molecule
in the presence of Ang-1 and as an agonistic molecule in the absence of
Ang-1. Experiments with the spheroidal EC/SMC model are now under way
to test this hypothesis.
Taken together, the experiments in this study have established an EC/SMC coculture system as an in vitro representation of the physiological assembly of a normal blood vessel that offers a unique experimental system for the analysis of paracrine interactions of EC and SMC. We have applied the coculture model toward an analysis of factors that control the quiescent EC phenotype to demonstrate that SMC regulate EC quiescence and control the responsiveness to angiogenic cytokines such as VEGF. Further analysis of the observations in this study will be critical to understanding the interplay of cellular and molecular regulators of blood vessel assembly, maintenance, and maturation. The versatility of the coculture model also suggests that it may be a powerful tool toward the analysis of other EC and SMC interactions that are critical regulators not just during vascular morphogenesis, but also in pathological processes such as restenosis and atherosclerosis.
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ACKNOWLEDGMENTS |
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Received for publication April 20, 2000.
Revision received August 1, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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