Journal of Cell Science 112, 3249-3258 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0536
3249
Tensional forces in fibrillar extracellular matrices control directional capillary
sprouting
Thomas Korff and Hellmut G. Augustin*
Cell Biology Laboratory, Department of Gynecology and Obstetrics, University of Göttingen Medical School, 37075 Göttingen,
Germany
*Author for correspondence (e-mail: [email protected])
Accepted 28 July; published on WWW 22 September 1999
SUMMARY
During angiogenesis, anastomosing capillary sprouts
align to form complex three-dimensional networks of new
blood vessels. Using an endothelial cell spheroid model
that was developed to study endothelial cell
differentiation processes, we have devised a novel collagen
gel-based three-dimensional in vitro angiogenesis assay.
In this assay, cell number-defined, gel-embedded
endothelial cell spheroids act as a cellular delivery device,
which serves as a focal starting point for the sprouting of
lumenized capillary-like structures that can be induced to
form complex anastomosing networks. Formation of
capillary anastomoses is associated with tensional
remodeling of the collagen matrix and directional
sprouting of outgrowing capillaries towards each other. To
analyze whether directional sprouting is dependent on
cytokine gradients or on endothelial cell-derived
tractional forces transduced through the extracellular
matrix, we designed a matrix tension generator that
enables the application of defined tensional forces on the
extracellular matrix. Using this matrix tension generator,
causal evidence is presented that tensional forces on a
fibrillar extracellular matrix such as type I collagen, but
not fibrin, are sufficient to guide directional outgrowth of
endothelial cells. RGD peptides but not control RAD
peptides disrupted the integrity of sprouting capillary-like
structures and induced detachment of outgrowing
endothelial cells cultured on top of collagen gels, but did
not inhibit primary outgrowth of endothelial cells. The
data establish the endothelial cell spheroid-based threedimensional angiogenesis technique as a standardized,
highly reproducible quantitative assay for in vitro
angiogenesis studies and demonstrate that integrindependent matrix tensional forces control directional
capillary sprouting and network formation.
INTRODUCTION
and, thus, contribute to providing a provisional matrix along
which sprouting capillaries continue to grow (Sephel et al.,
1996; Haralabopoulos et al., 1994; Ingber et al., 1986).
A number of assays have been established to study
angiogenesis and the properties of angiogenic endothelial
cells in culture. Two-dimensional lateral sheet migration and
proliferation assays have been used widely to study
angiogenesis in vitro (Kim et al., 1998; Augustin and Pauli,
1992; Pepper et al., 1989; Sato and Rifkin, 1988).
Reductionist as these assays may be, they clearly reflect some
aspects of the angiogenic cascade, as indicated by the fact
that several inhibitors of angiogenesis have been identified
using migration and proliferation inhibition assays as
screening systems (e.g. Ingber et al., 1990). Two-dimensional
assays have been established based on the observation that
endothelial cells cultured as monolayers on collagen gels or
Matrigel align to form a network of endothelial cell cords
(Vernon et al., 1992; Grant et al., 1989; Kubota et al., 1988;
Montesano et al., 1983). This cord-forming process reflects
morphogenetic properties of endothelial cells and has been
Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, is characterized by a complex
morphogenetic cascade of events during which quiescent
resting endothelial cells become activated to proteolytically
degrade their underlying extracellular matrix, directionally
migrate towards the angiogenic stimulus, proliferate and align
into new three-dimensional capillary networks (Augustin,
1998; Risau, 1997). The complexity of the angiogenic cascade
requires endothelial cells to perform a number of distinct
microenvironmental interactions with their surrounding
extracellular matrix. As a consequence, angiogenic endothelial
cells have a distinct gene expression pattern that is
characterized by a switch of the cells’ proteolytic balance
towards an invasive phenotype as well as the expression of
specific adhesion molecules such as the integrin heterodimers
αvβ3 and αvβ5 (Augustin, 1998; Bischoff, 1997; Preissner
et al., 1997). Furthermore, angiogenic endothelial cells
themselves synthesize components of the extracellular matrix
Key words: Angiogenesis, Endothelium, Spheroid, Extracellular
matrix
3250 T. Korff and H. G. Augustin
successfully used to study endothelial cell and extracellular
matrix interactions. As an angiogenesis assay, however, the
Matrigel assay appears to be of limited use, because cord
formation on Matrigel is not limited to only endothelial cells;
it has also been observed in nonendothelial cells (Vernon et
al., 1992).
In addition to planar assays, a number of three-dimensional
cell culture systems have been developed to study specific
steps of the angiogenic cascade. These include the capillary
invasion of a collagen or fibrin gel from a monolayer of
endothelial cells cultured on top of the gel (Montesano and
Orci, 1985), the overlay of an endothelial cell monolayer
cultured on top of a collagen or fibrin gel by another collagen
or fibrin gel (Chalupowicz et al., 1995), the outgrowth of
capillaries from an aortic ring embedded in a gel (Nicosia and
Ottinetti, 1990), and the sprouting of endothelial cells from
freshly isolated, collagen-embedded microvessels (Hoying et
al., 1996). Some studies have described the formation of
capillary networks originating from single gel-embedded
endothelial cells (Ment et al., 1997; Madri et al, 1988). In
these experiments, a large number of cells (1-2×106 cell/ml
gel) need to be seeded in the gel, suggesting that this
approach may be suitable for the analysis of capillary
alignment and remodeling studies, but does not really reflect
sprouting angiogenesis. More recently, a number of
experimental systems have been described that are aimed at
focally delivering aggregates of endothelial cells from which
sprouting can occur. Pepper et al. (1991) first described an in
vitro angiogenesis assay in which aggregates of endothelial
cells are embedded in collagen or fibrin gels. This assay has
been modified to deliver endothelial cells in fibrin gels by
growing them on microcarrier beads (Nehls and Drenckhahn,
1995) or by embedding endothelial cell aggregates in
collagen matrices, which are supported by annuli of nylon
mesh (Vernon and Sage, 1999).
We have recently described a three-dimensional spheroid
model of endothelial cell differentiation (Korff and Augustin,
1998). This study revealed that single nonadherent endothelial
cells are destined to undergo apoptosis and that they are not
responsive to the activities of cytokines that act as survival
factors. In contrast, spheroidal aggregation stabilizes
endothelial cells and renders them responsive to survival
factors (Korff and Augustin, 1998). These observations most
likely account for the fact that the embedding of single
suspended endothelial cells in collagen or fibrin gels leads to
massive apoptosis (Pollman et al., 1999; Satake et al., 1998; T.
Korff and H. G. Augustin, unpublished observations), whereas
the embedding of cellular aggregates or microcarrier adherent
cells leads to radial capillary sprouting. We therefore
conducted experiments aimed at developing an endothelial cell
(EC) spheroid-based in vitro angiogenesis assay that would be
a simple, highly reproducible, three-dimensional in vitro
angiogenesis assay. During these experiments we observed that
sprouting capillary-like structures grow directionally towards
each other when embedded in collagen gels. We hypothesized
that endothelial cell-derived matrix-transduced tractional
forces are responsible for this directionality effect and set up
experiments to investigate whether tensional forces are
sufficient to induce directional capillary sprouting or if
paracrine cytokine signaling phenomena are responsible for
this effect.
MATERIALS AND METHODS
Antibodies, growth factors and reagents
FGF-2 was obtained from Promega (Mannheim, Germany). VEGF
was from Upstate Biotechnology (Lake Placid, NY).
Carboxymethylcellulose (4.000 centipoises) and thrombin (bovine
plasma) were from Sigma (Deisenhofen, Germany). Fibrinogen
(bovine Plasma, clottable P>95%) was purchased from Calbiochem
(Bad Soden, Germany). RGD-containing peptides (GRGDSP) as well
as control RAD-peptides (GRADSP) were from Biomol (Hamburg,
Germany).
Cell culture
Endothelial cell growth medium (ECGM) and endothelial cell growth
supplement (human umbilical vein endothelial cell culture) were
purchased from Promocell (Heidelberg, Germany). Dulbecco’s
modified Eagle’s medium (DMEM) and other cell culture media were
from Life Technologies (Gibco BRL, Eggenstein, Germany). Fetal
calf serum (FCS) was obtained from Biochrom (Berlin, Germany).
Bovine aortic endothelial (BAE) cells were isolated from thoracic
aortas of healthy cattle by collagenase digestion following standard
protocols. Cells were cultured at 37°C in 75-cm2 tissue culture dishes
in DMEM containing 10% heat-inactivated fetal calf serum and frozen
in liquid nitrogen at passage 2 or 3. Cells were routinely split in a 1:5
ratio and cultured for up to 50 passages. Only BAE cells cultured from
passage 15-30 were used for experiments. Human umbilical vein
endothelial (HUVE) cells were freshly isolated from human umbilical
veins of newborn babies by collagenase digestion. Cells were cultured
at 37°C in 75-cm2 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 4-8 were used
for experiments.
Generation of endothelial spheroids
Endothelial cell spheroids of defined cell number were generated as
described previously (Korff and Augustin, 1998). In order to generate
endothelial cell spheroids of defined size and cell number, a specific
number of BAE or HUVE cells (750) were suspended in
corresponding culture medium containing 0.25% (w/v)
carboxymethylcellulose and seeded in nonadherent round-bottom 96well plates (Greiner, Frickenhausen, Germany). Under these
conditions all suspended cells contribute to the formation of a single
EC spheroid. These standardized spheroids were harvested within
24 hours and used for the corresponding experiments.
In vitro angiogenesis assay
In vitro angiogenesis in collagen gels was quantified using spheroids
of endothelial cells in a modification of the microcarrier-bead
angiogenesis assay (Nehls and Drenckhahn, 1995). In brief, HUVE
or BAE cell spheroids, containing 750 cells, were generated overnight,
after which they were embedded into collagen gels. A collagen stock
solution was prepared prior to use by mixing acidic collagen extract
of rat tails (equilibrated to 2 mg/ml, 4°C; 8 vol.) with 10× EBSS
(Gibco BRL, Eggenstein, Germany; 1 vol.) and 0.1 N NaOH (approx.
1 vol.) to adjust the pH to 7.4. This stock solution (0.5 ml) was mixed
with 0.5 ml room temperature medium [BAE cells: DMEM with 20%
FCS; HUVE cells: ECGM basal medium (PromoCell, Heidelberg,
Germany) with 40% FCS (Biochrom, Berlin, Germany)] containing
0.5% (w/v) carboxymethylcellulose to prevent sedimentation of
spheroids prior to polymerization of the collagen gel, 50-100 HUVE
or BAE cell spheroids, and the corresponding test substance. The
spheroid-containing gel was rapidly transferred into prewarmed 24well plates and allowed to polymerize (for 1 minute), after which 0.15
ml ECGM basal medium was pipetted on top of the gel. The gels were
incubated at 37°C in 5% CO2 at 100% humidity.
To generate spheroid-containing fibrin gels, BAE cell spheroids (750
cells/spheroid) were suspended in DMEM containing 10% FCS and
Directionality of capillary sprouting 3251
2.5 mg/ml fibrinogen. Polymerization was induced by the addition of
1 U thrombin/ml fibrinogen solution (Sigma, Deisenhofen, Germany)
after which the solution was rapidly mixed and transferred into 24-well
plates. When the gelation was finished (1 minute) 0.15 ml DMEM was
pipetted on top of the gel.
Two different techniques were applied to quantify in-gel
angiogenesis. For rapid screening experiments, it proved to be
sufficient to measure the length of the three longest capillary-like
sprouts that had grown out of each spheroid after 3 days (ocular grid
at 100× magnification), analyzing at least 10 spheroids per
experimental group and experiment. For a more detailed quantitative
analysis of in-gel angiogenesis, the cumulative length of all capillarylike sprouts originating from the central plain of an individual
spheroid was measured at 100× magnification using a digitized
imaging system (DP-Soft, Olympus) connected to an inverted
microscope (IX50, Olympus). Again, at least 10 spheroids per
experimental group and experiment were analyzed. This analysis
takes into consideration that the angiogenic response induced by a
specific substance is more appropriately reflected by the length of
individual capillary-like sprouts as well as the number of capillarylike sprouts. Both quantitation techniques give similar results. The
cumulative measurement of all capillary-like sprouts, however, has a
higher level of resolution that permits assessment of smaller
differences than the crude measurement of the three longest
capillary-like sprouts.
Directional endothelial cell migration assay
Defined tensional forces on an underlying collagen or fibrin matrix
were applied with a specialized mechanical device that served as a
matrix tension generator (Fig. 1). The matrix tension generator was
designed and built in cooperation with the Laboratory of Medical
Mechanics at the University of Göttingen. For experiments with the
matrix tension generator, a collagen or fibrin gel (1.0 ml) is poured
into one well of a 24-well plate and allowed to polymerize. The matrix
tension generator is inserted into the plate as shown in Fig. 1. The
device holds two needles, which are fixed into the gel 6.0 mm from
each other. One of the needles is laterally moved with a screw that
drives a calibrated thread. By moving the needle 1.5 mm, 2.0 mm or
3.0 mm, the gel is stretched by 25%, 33% or 50%, respectively. Next,
a single BAE cell spheroid was placed onto the stretched gel between
the two needles. The spheroid was allowed to adhere, after which the
cells grew out radially. Radial outgrowth was quantified
microscopically after 24 hours. To quantify directionality of
endothelial cell outgrowth as a consequence of matrix-transduced
tensional forces, the ratio of length to width of endothelial cell
outgrowth (directionality index) was calculated.
Morphological analysis
For morphological analysis, collagen or fibrin gels were fixed for 24
hours in HBSS containing 4% formaldehyde, after which they were
processed for paraffin embedding. Following dehydration (in a
graded series of ethanol and isopropanol, 24 hours each; 4°C), the
gels were immersed with paraffin I (melting temperature 42°C) for
24 hours at 60°C and paraffin II (melting temperature 56°C) for 36
hours at 70°C. Finally, the resulting paraffin block was cooled to
room temperature and trimmed for sectioning. Sections were stained
with Hematoxylin.
Ultrastructural analysis of fibrin and collagen gels
Collagen or fibrin gels (24 hours after polymerization) were cut into
pieces, washed in phosphate buffer (0.1 M, pH 7.4) and fixed 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
(50-80 nm) sections were cut, collected on copper grids, and
automatically stained with uranyl acetate and lead citrate for
observation with a Zeiss EM 10 electron microscope.
Fig. 1. Design of the matrix tension generator used for the
mechanical exertion of defined tensional forces on collagen and
fibrin gels. (A) The matrix tension generator fits into a 24-well plate
(1) and holds two needles in place (2), which are inserted in a
collagen or fibrin gel. The needle holders (3) are flexibly inserted
into the device and their heights can be adjusted manually (4). The
screw on the right (5) drives a calibrated thread through which the
needle holder of the right needle can be moved laterally to transmit
tensional stress onto the gel. (B) Lateral view of the matrix tension
generator showing a close up of the lateral needle moving device.
RESULTS
Endothelial cell spheroid based in vitro
angiogenesis assay
In order to explore the suitability of EC spheroids as focal
starting points for in-gel-based three-dimensional in vitro
angiogenesis experiments, EC spheroids of defined cell
number (750 cells/spheroid) were seeded in collagen gels and
the outgrowth of capillary-like structures was assessed
qualitatively and quantitatively. Endothelial cells originating
from the embedded spheroids invade the gel to form complex
networks of capillary-like structures (Fig. 2A). Cross sections
of collagen gels revealed numerous capillary-like structures
that differentiate to form a true capillary lumen (Fig. 2B-E).
These lumenized structures are lined by a single layer of
flattened endothelial cells. Occasionally, unicellular sprouts
originating from these lumenized tubes can be identified in the
gel (Fig. 2D). Cells that fail to integrate into the endothelial
monolayer exhibit a condensed and fragmented nuclear
morphology indicative of their programmed cell death
(apoptosis) (Fig. 2E).
Quantitative assessment of three-dimensional in vitro
angiogenesis was performed by microscopically measuring the
length of outgrowing capillary sprouts with an ocular grid (Fig.
3). Collagen gel-embedded HUVE cell spheroids had a low
3252 T. Korff and H. G. Augustin
level of spontaneous angiogenesis. Even in the presence of
20% serum, on average they only gave rise to few capillary
sprouts with less than 50 µm length over a 3-day period (Fig.
A
3A,B). This behavior reflected the low autocrine activity of
HUVE cells. HUVE cells were, however, readily responsive to
exogenous stimulation by angiogenic growth factors. Addition
of either FGF-2 or VEGF induced capillary sprouting of
HUVE cells (Fig. 3A,C,D). In contrast to the low spontaneous
angiogenic activity of HUVE cells, spheroids of BAE cells
gave rise to intense sprouting, resulting in the formation of up
to 300 µm long capillary-like structures within 3 days (Fig.
3E,F). In fact, even a reduction of serum concentrations to
2.0% did not significantly affect the high baseline angiogenic
activity of BAE cells (data not shown), indicating and
extending previous findings in different bioassays (Korff and
Augustin, 1998; Villaschi and Nicosia, 1993; Mignatti et al.,
1991) that BAE cells are strongly regulated by autocrine
activity. Addition of exogenous FGF-2 or VEGF stimulated
capillary sprouting of BAE cells, albeit to a much lower degree
compared with HUVE cells (Fig. 3E,G,H).
Capillary sprouting from gel-embedded EC
spheroids leads to directional outgrowth of
anastomosing capillaries
When seeding EC spheroids at different densities in the
collagen gel, we observed that beyond a critical spheroid
density, capillary sprouts originating from the spheroids gave
rise to complex anastomosing capillary-like networks (Fig.
2A). This observation prompted us to study systematically
directional sprouting in three-dimensional collagen gels and
the mechanisms that are responsible for this directionality.
Upon seeding of EC spheroids into collagen gels, capillary
sprouts grow radially in all three dimensions (Fig. 4A,B). After
about 2 days in the gel, some sprouts change their direction to
grow towards a neighboring spheroid if this spheroid is in close
proximity. Analysis of a large number of gel-embedded
spheroids indicated that endothelial cell sprouts can sense
directionality in collagen gels over a distance of approximately
600-800 µm (Fig. 4C-E). Eventually, as the distance between
different capillary sprouts becomes smaller, several sprouts
align to span the gel between the two neighboring spheroids
(Fig. 4F). The directionality effect was identified as a specific
phenomenon of collagen matrices, since no signs of directional
capillary sprouting were observed in fibrin gels (data not
shown).
Fig. 2. Lumenized capillary sprouts originating from collagen gelembedded spheroids of BAE cells. Gel-embedded spheroids give rise
to radially outgrowing capillary sprouts. Outgrowing sprouts of
neighboring spheroids grow directionally towards each other to
establish networks of anastomosing capillary-like structures, as
shown by phase-contrast microscopic analysis of three adjacent
spheroids (A). Cross sections of gels with sprouts originating from
EC spheroids show capillary-like structures of varying size with
lining endothelial cells that form a true lumen throughout the gel.
The morphological appearance of these capillary sprouts ranges from
very small vessels lined by few EC (B-C) to larger structures lined by
numerous EC that form a bigger lumen (D-E). Depending on the
plain of section, sprouting EC originating from lumenized capillary
sprouts can be identified (D, arrow). Integration of EC into the lining
monocellular surface layer stabilizes the cells. Cells that are not
integrated into the monolayer become apoptotic (E, arrows). Bars,
500 µm (A); 20 µm (B-E).
Outgrowing capillary sprouts exert tractional forces
on the extracellular matrix
When spheroid-derived sprouts invade the collagen, they exert
tractional forces onto the gel. These tractional forces are
responsible for the shrinkage effect frequently observed when
cells are embedded in floating collagen gels (Vernon and Sage,
1996; Gullberg et al., 1990; Harris et al., 1981). In the present
study, we used nonfloating collagen gels that were solidly
anchored in their tissue culture well. Nevertheless, regional
differences in tractional forces could be identified when
observing neighboring spheroids over several days. Fig. 4A
shows two collagen gel-embedded neighboring spheroids
approximately 1.6 mm apart. After 5 days in the collagen gel
and extensive directional sprouting, the centers of the two
spheroids were only approximately 1.2 mm apart, indicating
that the two spheroids have moved 25% closer together.
Based on this observation, we analyzed the fibrillar structure
of the collagen gels that contained capillary-like structures.
Directionality of capillary sprouting 3253
The collagen fibrils between two neighboring spheroids were
found to align along the axis that connects the two spheroids
(Fig. 5A,C). The distance over which this alignment was found
corresponded to the distance up to which we observed
directional capillary sprouting (500-700 µm). When the
sprouts come into even closer proximity (<200 µm and less),
essentially all collagen fibrils between the sprouts are aligned
to connect the tips of the two adjacent sprouts.
Fig. 3. Quantitative three-dimensional in vitro angiogenesis assay based on collagen gel-embedded endothelial cell spheroids. Capillary
sprouting originating from the spheroids was quantified with a digitized imaging system as described in Materials and Methods, using human
umbilical vein endothelial (HUVE) cells (A) and bovine aortic endothelial (BAE) cells (E). Representatives of each experimental group are
shown in (B-D) (HUVE cells) and F-H (BAE cells). (B) Control HUVE cells; (C) HUVE cells + FGF-2 (30 ng/ml); (D) HUVE cells + VEGF
(50 ng/ml); (F) control BAE cells; (G) BAE cells + FGF-2 (30 ng/ml); (H) BAE cells + VEGF (50 ng/ml). HUVE cells have a low baseline
level of capillary sprouting and respond strongly to exogenous FGF-2 and VEGF. In contrast, BAE cells have a two times higher baseline
sprouting activity, reflecting a much higher degree of autocrine activity (***, P<0.001 compared to corresponding control). Bars, 150 µm.
Fig. 4. Directional sprouting of capillary-like structures towards each other, originating from two neighboring gel-embedded spheroids with a
distance of approximately 1.6 mm (measured from the center of each spheroid, A). Sprouts grow radially out of the spheroid for the first 2 days.
After 3 days, capillary sprouts start to change their direction to grow towards each other, which becomes even more evident after 4 and 5 days.
Note that the centers of the two spheroids have moved closer together after 4 and 5 days, reflecting the tractional forces exerted by the
outgrowing endothelial cells. Bar, 200 µm.
3254 T. Korff and H. G. Augustin
Fig. 5. (A,B) Cross sections of Hematoxylin-stained collagen gels
containing capillary-like structures at different distances from each
other (approx. 400 µm, A; approx. 70 µm, B). (C, D) Schematic
representations of the alignment of collagen fibrils shown in A and
B, respectively. Tractional forces exerted by sprouting endothelial
cells lead to an alignment of collagen fibrils, which can be observed
up to distances of 500-700 µm (A,C). When the capillary sprouts get
into closer proximity, essentially all of the collagen fibrils between
the sprouts have aligned (B,D). Bars, 100 µm (A); 20 µm (B).
Matrix-transduced tensional forces control
directional outgrowth of endothelial cells in collagen
gels but not in fibrin gels
The observations made thus far indicate that (1) invading
endothelial cells exert tractional forces on the matrix, (2)
collagen fibrils align along the axis of the matrix tension, and
(3) capillary sprouts grow directionally along these aligned
collagen fibrils to form anastomosing capillary-like networks.
We next set up experiments to investigate whether matrixtransduced tensional forces exerted by invading endothelial
cells are responsible and sufficient for directional endothelial
cell outgrowth. In order to exclude possible paracrine cytokine
signaling effects of endothelial cells sprouting from two
neighboring spheroids, we performed experiments with single
spheroids, using a mechanical device that served as a matrixtension generator (Fig. 1). By controlling the lateral movement
of two needles that are inserted into a collagen or fibrin gel,
this device can be used to apply defined tensional forces onto
the gel (Fig. 6A). After applying tensional stress onto the gel,
a single EC spheroid is seeded on the gel between the two
needles. If the applied tension is sufficient to control
directional outgrowth of the cells, this should lead to a
nonradial, ellipsoid pattern of outgrowth along the axis of the
tensional stress, which can be used to calculate a directionality
index (Fig. 6B). Additionally, this experiment would
demonstrate that outgrowing cells do not just exert tractional
forces onto the extracellular matrix, but rather that they can
‘read’ the direction of tension-aligned fibrils in the matrix.
As shown in Fig. 6C, a perfectly radial outgrowth of
endothelial cells was observed, when a single EC spheroid was
seeded on top of a nonstretched collagen gel. With increasing
tensional stress, outgrowth of endothelial cells became more
asymmetric along the direction of the tensional forces exerted
Fig. 6. Analysis of directional endothelial cell outgrowth by
mechanical manipulation of matrix tension with a matrix-tension
generator. (A) Conceptual design of the matrix-tension generator.
Two needles are inserted into a collagen or fibrin gel and controlled
tensional forces are exerted by lateral movement of one of the
needles, after which a single EC spheroid is seeded between the
needles (for details compare Fig. 1 and Materials and Methods).
(B) Quantification of a directionality index. The length and the width
of the front of outgrowing endothelial cells is measured
microscopically and a directionality index calculated as the ratio of
length to width. (C) Radial outgrowth of BAE cells from a single
spheroid seeded on top of a nonstretched collagen gel.
(D) Directional outgrowth of BAE cells from a single spheroid
seeded on top of a stretched collagen gel. (E) Quantification of
directional endothelial cell outgrowth after seeding of a single BAE
cell spheroid on top of a stretched collagen or fibrin gel. There is
significant, dose-dependent directional outgrowth of BAE cells
grown on top of a stretched collagen gel (P<0.001 at all points
compared to control). In contrast, stretched fibrin gels do not support
directional outgrowth. Bar, 100 µm.
by the matrix tension generator (Fig. 6D), leading to a dosedependent increase of the directionality index (Fig. 6E). In
contrast to the directional outgrowth of endothelial cells
cultured on top of stretched collagen gels, no such
Directionality of capillary sprouting 3255
directionality was observed when EC spheroids were placed on
top of fibrin gels. Even when the fibrin gels were prestretched
by 50%, no significant directional outgrowth of endothelial
cells was observed (Fig. 6E), confirming that fibrin gels do not
support directional capillary sprouting in three-dimensional
gels.
In order to further analyze the difference between collagen
and fibrin gels in supporting directional endothelial cell
outgrowth, collagen and fibrin gels were ultrastructurally
analyzed by electron microscopy. Analysis of in vitro
polymerized collagen confirmed the complex structure of
collagen gels with twisted, irregularly assembled collagen
fibrils several µm long, which lack the regular striated
appearance of in vivo assembled collagen fibrils (Fig. 7A)
(Fratzl et al., 1997; Ploetz et al., 1991). In contrast, analysis of
in vitro polymerized fibrin revealed the compact, densely
meshed structure of fibrin gels with short fibrils only a few
hundred nm long.
Integrin-mediated cell matrix contacts are involved
in regulating three-dimensional capillary sprouting
and two-dimensional EC outgrowth
The tractional forces exerted by invading endothelial cells on
the surrounding extracellular matrix involve complex adhesive
interactions between the cells and the matrix. In order to
Fig. 8. Effect of RGD peptides on in vitro angiogenesis. BAE cell
spheroids were either embedded in collagen gels (A,C) or cultured
on top of collagen gels (B,D) and incubated for 3 days in the
presence of 30 µM RGD peptides or with the same concentration of
control RAD peptides. (A) Integrin-dependent contacts inhibiting
RGD peptides disrupt capillary-like sprouting in collagen gels.
(B) Addition of RGD peptides to laterally migrating endothelial cells
from a spheroid cultured on top of a collagen gel leads to rounding
and detachment of the cells. (C,D) Control RAD peptides have no
effect on in gel angiogenesis (C) or on cellular outgrowth on top of a
collagen gel (D). Bars, 100 µm.
analyze the contribution of integrin-mediated cell matrix
contacts on sprouting and directional endothelial cell
migration, three-dimensional in vitro angiogenesis
experiments and two-dimensional on-gel cellular outgrowth
experiments were performed in the presence of RGD or control
RAD peptides. A concentration of 30 µM RGD disrupted
capillary morphogenesis in three-dimensional collagen gels
(Fig. 8A). Interestingly, invasion of EC into the gel was not
inhibited by RGD peptides, but addition of the peptide
disrupted the integrity of the capillary sprouts. On twodimensional collagen gels, addition of RGD peptides induced
the rounding and detachment of the cells from the matrix, but
corresponding to the experiments in three-dimensional gels,
they did not inhibit cellular outgrowth as such (Fig. 8B). In all
of these experiments, RAD peptides (30 µM) served as
controls. RAD peptides neither inhibited in vitro angiogenesis
in three-dimensional collagen gels (Fig. 8C) nor affected EC
outgrowth or monolayer integrity on top of collagen gels (Fig.
8D).
DISCUSSION
Fig. 7. Ultrastructural analysis of a collagen (A) and a fibrin (B) gel.
(A) Longitudinal and transverse section of a collagen gel fibrils.
Collagen forms twisted fibrils several µm long. (B) In contrast, fibrin
fibrils are short and have a compact, densely meshed structure. Bar,
200 nm.
The complexity of the angiogenic cascade is
recognized. Originally supported by a simplified
invasive, migratory, proliferating, angiogenic
cell, angiogenesis is now widely viewed as
increasingly
model of an
endothelial
a complex
3256 T. Korff and H. G. Augustin
morphogenetic event that includes discrete steps of capillary
organization, tubular branching, network formation and vessel
maturation (Darland and D’Amore, 1999; Hanahan, 1997),
leading to the development of in vitro assays of angiogenesis
to facilitate the study of individual steps of the angiogenic
cascade under defined conditions. Towards this end a number
of two-dimensional and three-dimensional assays have been
developed (Vernon and Sage, 1999; Hoying et al., 1996; Nehls
and Drenckhahn, 1995; Augustin and Pauli, 1992; Nicosia and
Ottinetti, 1990; Grant et al., 1989; Madri et al, 1988;
Montesano and Orci, 1985; Montesano et al., 1983) and all of
these assays legitimately reflect some aspects of angiogenesis.
Nevertheless, the limitations of in vitro systems necessitate
caution when interpreting findings from such in vitro models
to the in vivo situation (Passaniti, 1992; Madri and Basson,
1992; Grant et al., 1992; Vernon and Sage, 1992)
EC spheroid-based three-dimensional in vitro
angiogenesis assay
The aim of the present study was to develop a novel threedimensional in vitro angiogenesis assay, taking advantage of a
recently established spheroid model of endothelial cell
differentiation (Korff and Augustin, 1998). EC spheroids can
be produced at any size with a defined number of cells. The
EC spheroid-based in vitro angiogenesis assay proved to be
simple and highly reproducible. The assay has a number of
advantages over other EC aggregate-based in vitro
angiogenesis assays. By utilizing cell number-defined EC
spheroids, standardized experimental conditions can be
applied. In comparison to the microcarrier bead angiogenesis
assay (Nehls and Drenckhahn, 1995), larger numbers of
endothelial cells can be focally applied into the gel, avoiding
the possibility that EC proliferation may become rate-limiting
for the sprouting angiogenesis process.
The very distinct advantage of all cell aggregate-based
angiogenesis assays is the fact that endothelial cells can be
focally delivered into a three-dimensional matrix, allowing
sprouting angiogenesis to occur by invasion into the
extracellular matrix. This process is very much in contrast to
the alignment of large numbers of gel-embedded single
endothelial cells and appears to be the closest in vitro
representation of the angiogenic invasion of the extracellular
matrix as it occurs during angiogenesis in vivo. If sprouting
angiogenesis originating from gel-embedded EC spheroids is
allowed to proceed for several days, capillary sprouts lead to
the formation of complex three-dimensional networks, which
can be used to analyze individual steps of the angiogenic
cascade sequentially and the interactions of the forming
endothelial cell network with mural cells (T. Korff and H. G.
Augustin, manuscript in preparation).
We have applied the EC spheroid-based in vitro
angiogenesis assay towards the analysis of the angiogenic
capacity of two different cell populations, human umbilical
vein endothelial (HUVE) cells and bovine aortic endothelial
(BAE) cells. The two cell populations show distinct differences
in their angiogenic capabilities, making them suitable target
cell populations for different specific questions. HUVE cells
have a low baseline angiogenic activity requiring stimulation
with exogenous angiogenic cytokines such as VEGF or FGF2 for angiogenesis to occur. This makes them suitable target
cells for the analysis of angiogenesis-promoting substances.
We recently exploited this capacity in the characterization of
the newly identified Orf virus-encoded VEGF variant VEGFE (Meyer et al., 1999). In contrast, BAE cells have a high
baseline angiogenic activity in the absence of exogenous
cytokines, even under strongly serum-reduced culture
conditions. This high degree of autocrine activity corresponds
to the high intensity of autocrine activity of these cells that has
been observed in a number of other bioassays and appears to
be largely mediated by endogenous expression of FGF-2 (Korff
and Augustin, 1998; Villaschi and Nicosia, 1993; Mignatti et
al., 1991). As a consequence, the autocrine activity makes BAE
cells highly suitable for the study of angiogenesis-inhibiting
substances.
Directional capillary sprouting in collagen versus
fibrin gels
Gel-embedded EC spheroids gave rise to complex capillarylike networks. This network-forming activity appears not to be
a random process, but rather the consequence of directional
outgrowth of capillary sprouts towards each other. Directional
capillary sprouting appears to be a critical morphogenetic
determinant during vessel formation in vivo. During
vasculogenesis, a primitive capillary plexus is formed as a
consequence of the tractional coalescence of in situ
differentiating angioblastic cells (Risau and Flamme, 1995).
Likewise during angiogenesis, capillary sprouts form
anastomoses that give rise to capillary networks (Benjamin et
al., 1998).
Capillary-like network formation has been associated with
mechanical forces exerted by angiogenic endothelial cells onto
its surrounding extracellular matrix (Vernon and Sage, 1995;
Ingber and Folkman, 1989). Alternatively, or acting in concert
with mechanical forces, paracrine cytokine signaling processes
may be responsible for the directed growth of two capillary
sprouts towards each other. Tractional forces of cells exerted
onto its surrounding fibrillar extracellular matrix have been
most extensively studied in fibroblasts. Fibroblast-mediated
tractional forces are believed to be primary morphogenetic
determinants in the formation of tendons as well as during
wound contraction (Harris et al., 1981). Endothelial cells have
been reported to exert similar tractional forces on type I
collagen as dermal fibroblasts (Vernon and Sage, 1996).
Analysis in planar two-dimensional systems has given good
evidence that mechanical forces exerted by endothelial cells
actively modulate the underlying extracellular matrix and that
this matrix modulatory effect may contribute to capillary
morphogenesis (Vernon et al., 1992). Most of these
experiments were performed with Matrigel as extracellular
matrix. Matrigel is rich in laminin, type IV collagen and
fibronectin and may not be very representative of the type I
collagen-rich interstitial matrix that endothelial cells are
mostly exposed to during physiological angiogenesis in vivo.
Correspondingly, planar tube formation assays have similarly
shown that endothelial cell-derived tractional forces can
modulate the directional alignment of individual collagen
fibrils (Vernon and Sage, 1995).
In the present study we observed that sprouts originating
focally from collagen gel-embedded EC spheroids give rise to
complex three-dimensional networks of directionality towards
each other, growing anastomosing, capillary-like structures.
Morphological analysis of the gels demonstrated that collagen
Directionality of capillary sprouting 3257
fibrils between adjacent capillary-like sprouts align over
distances of up to 1 mm. This distance can be reduced or
increased somewhat by changing the mechanical properties of
the gel, but it appears to correspond well with the geometrical
properties of experimental models of capillary network
formation in vivo, as can be observed, for example, during
rabbit cornea angiogenesis (Kozian et al., 1997) or postnatal
retinal vascularization (Benjamin et al., 1998).
It has long been speculated that matrix-transduced tensional
forces are responsible for directional capillary sprouting
(Vernon and Sage, 1995). To date, however, no causal evidence
has been presented to demonstrate that matrix-transduced
tensional forces are responsible and sufficient for this effect.
This prompted us to develop a mechanical device that is
capable of generating defined mechanical tension on to the
matrix and allowed us to study the outgrowth of endothelial
cells on top of stretched collagen and fibrin gels to exclude
paracrine cytokine-mediated signaling effects which might be
responsible for the directional capillary sprouting towards each
other. These experiments showed unambiguously for the first
time that matrix-transduced tensional forces in stretched
collagen gels are sufficient to control the directional outgrowth
of endothelial cells. In turn, the experiments also show that
matrix-transduced tensional forces are, thus, not just a
byproduct of tractional forces exerted by invading endothelial
cells, but rather that outgrowing endothelial cells can ‘read’ the
direction of tension-aligned fibrils in the extracellular matrix.
Directional capillary sprouting was only observed in type I
collagen gels, but not in fibrin gels. Likewise, directional
outgrowth on stretched gels was only seen in type I collagen
gels and not in fibrin gels. As confirmed by direct electron
microscopic analysis and corresponding well with data in
established literature, fibrin forms much shorter fibrils than
collagen, which appears to limit its mechanotransducing
capacity. The different behaviors of fibrin and collagen gels in
supporting directional capillary sprouting and network
formation may not merely reflect in vitro behavior of cells in
gels, but rather suggests some thought-provoking hypotheses
on the mechanisms of capillary network formation in vivo.
Physiological, developmental angiogenesis occurs mostly
through a collagen-rich extracellular matrix and leads to the
formation of a regular capillary network. In contrast,
pathological angiogenic processes in the adult, as they are
associated with tumor growth or wound healing, lead to the
formation of an irregular, highly tortuous capillary network, as
was shown by numerous casting experiments (Konerding et al.,
1995). Tumor and wound healing angiogenesis, however, are
capillary sprouting processes known to primarily occur in a
fibrin-rich matrix (Dvorak, 1986).
CONCLUSION
Taken together, the present study presents causal evidence that
matrix-transduced tensional forces are critical determinants in
capillary morphogenetic network formation processes. Matrixtransduced tensional forces are sufficient to control directional
EC outgrowth. Invasive endothelial cells do not just create
tractional forces on the matrix, but they can ‘read’ the tensionaligned orientation of fibrillar extracellular matrices.
Directional capillary sprouting processes are limited to large
fibrillar matrices such as type I collagen and are not observed
in short fibrillar matrices such as fibrin. This finding may have
implications for the differences in the capillary morphogenetic
processes that occur in collagen- and fibrin-rich matrices in
vivo, e.g. the differences observed between regular
developmental angiogenesis in a collagen-rich matrix and the
irregular, tortuous capillaries that form in fibrin matrices as
they are associated with pathological angiogenic processes
such as the growth of tumors or the healing of a wound.
Furthermore, the angiogenesis assay described in this study
may prove to be a versatile tool for quantitative and qualitative
angiogenesis studies in vitro. It is simple, highly reproducible,
easy to quantify, and allows the establishment of complex
three-dimensional networks in culture. This may prove useful
for studies aimed at sequentially analyzing individual steps of
the angiogenic cascade, including detailed morphogenetic
studies involving branchpoint analyses and the formation of
anastomoses as well as endothelial cell and mural cell
interactions.
The authors would like to acknowledge the excellent technical
assistance of Mrs Renate Dietrich and Mrs Cathleen Lakoma. We
thank Dr Franz-J. Kaup (DPZ, Göttingen, Germany) for assistance
with the electron microscopic analyses and Mr Wegener (Laboratory
for Medical Mechanics, University of Göttingen, Germany) for
support in designing and constructing the matrix tension generator.
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft (SFB500, C3).
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