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From the Department of Cardiovascular Research, Genentech Inc., South San Francisco, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various in vitro systems have been used to model the differentiation of endothelial cells into tubular capillary-like sprouts. The mechanism of tube formation is not clearly understood, and the different in vitro models may yield divergent information due to differences in the matrix components, growth factor requirements, and even the source of the endothelial cells used. A commonly used method of in vitro angiogenesis is the spontaneous or growth-factor-induced formation of cord-like structures when endothelial cells are cultured on an extracellular matrix secreted by Engelbroth-Holm-Swarm sarcoma (EHS) cells, commonly known as Matrigel. This model has been used by many investigators to demonstrate roles for various ligands, receptors, and integrins in the angiogenic process, and many of these findings have been verified by in vivo experiments. However, Matrigel-driven tube formation can occur independent of new protein or mRNA synthesis,1,2 although there are conflicting reports.3 Studies by Davis et al suggested that the cords that form in the Matrigel system follow linear distortions, so-called matrix guidance pathways that are generated in the matrix by the generation of mechanical tension between endothelial cells.4 Analysis of the structure of the cords formed also suggests that lumens are not generally found, and in the rare cases in which they have been described, basement membrane material is present on both apical and basal aspects.5-8 This probably occurs by enfolding of the supportive scaffold by tractional forces, resulting in the tubular shape.9 The lack of apical:basal polarity as well as the infrequent generation of patent lumens suggests that the Matrigel model may not be a suitable system to probe the molecular events that lead to lumen formation. Additionally, a variety of cell types will form networks on Matrigel, including certain types of epithelial cells, dermal fibroblasts, smooth muscle cells, osteoblasts, keratinocytes, renal tubular cells, and hepatocytes,9-15 clearly indicating that the formation of networks on Matrigel is not endothelial specific.
Endothelial cells will also differentiate in three-dimensional gels
generated from type I collagen or fibrin. A commonly used method is to
sandwich the endothelial cells between two layers of the matrix. Soon
after the sandwich is formed, the endothelial cells undergo
morphogenesis to form a structure resembling a capillary network that
is extensive and well formed within 24 hours16
with
infrequent but appropriately polarized luminal structures. Endothelial
cells grown on the surface of a collagen gel can be induced to invade
upon addition of phorbol myristate acetate (PMA)8
or
antibodies to 
2ß1 integrins.17
Under these conditions,
the majority of invading cells have vacuole-like structures and
multicellular lumens. A modification of the type I collagen gel model
incorporates endothelial cells into the collagen before gelation, and
after the gel has formed, culture medium containing PMA, basic
fibroblast growth factor (bFGF), and vascular endothelial cell growth
factor (VEGF) is added. The endothelial cells rapidly differentiate
forming tube-like structures with frequent lumens with appropriate
apical:basal polarity.18
In the present communication, we used the "In Gel" version of the type I collagen gel matrix18 to begin to dissect the molecules and events that lead to the formation of interconnected tubes and lumens. These studies demonstrate the obligatory role for new mRNA and protein synthesis in this differentiation as well as an important and possibly differential role for two endothelial junction proteins, platelet-endothelial adhesion molecule-1 (PECAM-1, CD31) and vascular-endothelial cadherin (VE-cadherin, CD144).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (San Diego, CA) and maintained in EGM media (Clonetics) supplemented to a final concentration of 10% fetal bovine serum (FBS). Type I rat tail collagen was from Upstate Biotechnology (Lake Success, NY) and recombinant bFGF was purchased from Collaborative Biomedical Products (Becton Dickinson Labware, Bedford, MA). Recombinant VEGF was from Genentech (South San Francisco, CA). 10X medium 199 (M199, M0650), PMA, 4,6'-diamino-2-phenylindole dihydrochloride (DAPI), actinomycin D, cycloheximide, phycoerythrin-conjugated goat anti-mouse IgG, and isotype-matched control (IgG1 and IgG2a) were from Sigma Chemical Co. (St. Louis, MO). Antibodies to CD31 were as follows: clone BV10 (Chemicon, Temecula, CA), clone JC/70A (DAKO, Glostrup, Denmark), clone HEC7 (Endogen, Woburn, MA); antibodies to VE-cadherin: clone RM67 (Chemicon) and TEA1/31 (Immunotech, Marseilles, France). Antibody to ICAM-1 (clone P2A4) was purchased from Chemicon. Rhodamine phalloidin was from Molecular Probes (Eugene, OR). FBS was from Hyclone (Logan, UT). ITS (insulin, transferrin, and selenium-A) and trypsin, and were from Gibco-BRL (Gaithersburg, MD).
Formation of Three-Dimensional Collagen Gels
Collagen gels were formed by mixing together ice-cold gelation solution (10X M199, H2O, 0.53 mol/L NaHCO3, 200 mmol/L L-glutamine, type I collagen, 0.1 mol/L NaOH; 100:27.7:50:10:750:62.5 by volume) and cells in 1X basal medium (see below) at a concentration of 3 x 106 cells/ml at a ratio of 4 vol of gelation solution:1 vol of cells. The gels were allowed to form by incubation in a CO2-free incubator at 37°C for 30 minutes to 1 hour. The gels were then overlaid with 1X basal medium consisting of M199 supplemented with 1% FBS, 1X ITS, 2 mmol/L L-glutamine, 50 µg/ml ascorbic acid, 26.5 mmol/L NaHCO3, 100 U/ml penicillin, 100 U/ml streptomycin and, unless otherwise noted, 40 ng/ml bFGF, 40 ng/ml VEGF, and 80 nmol/L PMA. In antibody-blocking and drug studies the substances were added to the 1X basal medium. At the end of the experiment the media were removed by gentle aspiration and the gels routinely fixed in 3.7% formalin. Cells were later stained with rhodamine phalloidin (5 U/ml) and DAPI (4 µmol/L final concentration) to visualize the actin cytoskeletons and nuclei, respectively. In some experiments the lumens were loaded with 6-carboxyfluorescein, single isomer (6-CF), by including the dye solution at a concentration of 0.06 mg/ml with the endothelial cells during the formation of tubes and lumens. Free dye was removed by repeated washing of fixed gels with PBS. In some experiments, fixed cells were stained 30 minutes with toluidine blue in Tris buffer (0.1 mol/L Tris/0.15 mol/L NaCl, pH 9.5). The gels were destained in a bath of Tris buffer (2 to 5 hours depending on size and thickness of gels). Gels were removed from the bath, rinsed once with Tris buffer, and then maintained in a PBS solution. Photographs were taken on a Nikon Eclipse TE300 inverted phase microscope using Hoffmann modulation contrast optics and a Polaroid digital microscope camera. Images were captured using Adobe Photoshop and composite photomicrographs prepared using QuarkExpress software.
Quantification of Tube Formation
Gels were examined on a Nikon TE300 microscope equipped with Hoffman modulation optics and a cooled CCD camera (Optronics, Technical Instruments, San Francisco, CA). Assays were performed using 48-well plates with n=3 wells for each experimental manipulation, and all experiments were repeated at least three times. The optimal optical quality of the 48-well chamber is in the center of each well, and this region was selected for image capture. After focusing on the lowest layer of the gel (ie, the bottom), the stage was moved upward 100 units on the coaxial fine focusing knob and the first image captured. Four additional fields, separated by 100-unit increments were imaged, and the data were imported as TIFF files into NIH Image. After calibration with a stage micrometer, a second observer measured the total length of each tube or the long axis of single cells or groups of adjacent cells that were in clear focus in the image field, and the data were imported into a Microsoft Excel spread sheet. In those instances where several tube-like structures merged together or branched, the total length of the tubes was calculated as the sum of the length of the individual branches. The number of tubes (or cells) containing one or more vacuoles/lumens was also collected for each image. The mean length and percentage of cells/tubes containing vacuoles was determined for each well, followed by determination of the mean data and standard deviation for each experimental group. In some experiments, the number of tube-like structures (defined as those exceeding 100 µm in length) per high-power (20x objective) field was also determined. Data were analyzed by one-way analysis of variance and post hoc Bonferonni's modified t-test for multiple comparisons. P < 0.05 was accepted as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As early as 2 hours, small vacuole-like structures could be
observed in select individual endothelial cells (~16% of the total
cell population). The frequency of vacuole formation increased such
that at 8 hours, virtually all cells in the image field contained these
vacuoles. As early as 4 hours, the smaller vacuoles appeared to fuse to
form large vacuole structures. At 48 hours, these structures appeared
as interconnecting lumens, formed either by individual cells aligned
end to end or, in some cases, by several cells surrounding a single
lumen. Although there was some variability from one endothelial cell
preparation in the time required to form branching tube-like structures
and in the mean length of the tubes at 48 hours, nearly every cell in
the gel was associated with a tube-like structure. Those that had not
been incorporated into a tube appeared to remain viable and exhibited
an elongated morphology and large intracellular vacuoles. The branching
structures continued to become more complex over the next 24 hours.
However, many preparations of HUVECs appeared to degrade the gels at
times approaching or exceeding 72 hours, complicating any longer-term
evaluation of tube formation or assessment of possible regression of
the tubes. Figure 1, A–I
, shows
representative images of the morphology observed at 4, 24, and 48
hours. Figure 2
provides quantitative
analysis of the mean tube length and the percentage of cells or
tube-like structures that contained vacuole-like structures.
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. Use of the acetoxy methyl
ester forms of 6-CF or other fluorescent dyes did not result in loading
of the luminal structures. Acetoxy methyl ester derivatives are
membrane permeable and are used to load the cytoplasm of endothelial
cells. The acetoxy methyl ester is cleaved by cell esterases and the
nonpermeant form of the dye becomes trapped in the cytoplasm. The
preferential loading of the free form of the dye is consistent with
uptake of the dye by vesicular transport of material from the
extracellular space.
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To determine the role for new protein and mRNA synthesis in the
processes of vacuole, tube, and lumen formation, at the time of
addition of the basal media supplemented with PMA, bFGF, and VEGF, 1
µg/ml actinomycin D or 10 µg/ml cycloheximide were added to the
cultures. Preliminary studies indicated that these concentrations of
drugs were sufficient to inhibit >95% of radiolabeled uridine or
leucine uptake, respectively. As shown in Figure 4, C and D
, cells co-incubated with
either actinomycin D or cycloheximide did not form interconnecting
sprouts or observable lumens. Cells treated with actinomycin D or
cycloheximide remained round and did not elongate when observed at
earlier time points (2, 4, 8, or 24 hrs (not shown)). As shown in
Figure 4E
, gels incubated with either actinomycin D or cycloheximide
demonstrated a marked reduction in the mean length of the structures
(basically the mean long axis of rounded cells), and these effects were
statistically significant.
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CD31 and VE-cadherin have been implicated in angiogenesis in
vitro using several different models.19-22
We
therefore evaluated the respective roles of these matrix and junctional
adherence23
molecules in the three-dimensional collagen gel
model. A variety of antibodies for each molecule were acquired from
commercial sources as detailed in Materials and Methods. As shown in
Figure 5A
, CD31 and VE-cadherin
antibodies significantly decreased the number of tube structures
(defined as contiguous structures exceeding 100 µm in length) per
high-power field in the three-dimensional collagen gel. The
corresponding isotype-matched nonimmune control antibodies had no
effect on endothelial tube formation. Additionally, an endothelial
binding antibody (anti-ICAM-1) also failed to inhibit tube formation
(Figure 5B)
. Some interesting morphological differences, in comparison
with controls, were found upon high-power examination of the structures
formed during incubation with the different antibodies. All of the CD31
antibodies tested attenuated the elongation of the cells into tube-like
structures (Figure 5
and Figure 6, C–F
).
Many of the cells were isolated and had a large intracellular vacuole
(Figure 6, E and F)
. Others tended to aggregate and form attenuated
(ie, shorter) tube-like structures with a large lumen (Figure 6, C and D)
. Both morphologies were observed with all three anti-CD31 antibodies
used, ie, BV10, JC/70A, and HEC7. In contrast, cells incubated with the
anti-cadherin antibody TEA 1/31 tended to elongate (although the
average length of the tubes formed was significantly shorter than those
observed in the absence of antibody, Figure 5B
) and to not associate
with adjacent cells, and furthermore, nearly one-half of these
elongated structures were devoid of vacuoles (Figure 6, I and J)
,
whereas others had only small attenuated vacuoles usually found at one
end of the elongated structure (Figure 6, G and H)
. However, cells
incubated in the presence of the anti-cadherin antibody RM67
demonstrated a reduction in mean length and in the number of
capillary-like structures (Figure 5, A and C)
, but the morphology was
distinct (Figure 6, K–N)
; ie, cells were somewhat elongated,
occasionally associated with adjacent cells, and the vacuoles of these
adjacent cells often were not fused (Figure 6, K and L)
. In
other instances isolated single cells could be visualized with
multiple, nonfused vacuoles (Figure 6, M and N)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial observations by time-lapse photography during the first 3 hours
after HUVECs were suspended in the three-dimensional gels indicated
that the endothelial cells displayed rapid, random extensions of fine
pseudopodia that eventually became longer (data not shown). At the same
time, the cells were forming numerous vacuoles that were merging to
form large intracellular vacuoles. In the presence of CD31 antibodies,
cell elongation, and apparently cell migration (as the cells appeared
isolated from one another) were blocked, but the vacuolization process
did not appear to be impaired. Engagement of CD31 has been reported to
activate ß1 and ß2 integrins in leukocytes and
platelets26-28
and could potentially modulate endothelial
integrins. CD31 has been shown to interact with integrin
vß3,29
an integrin with a demonstrated role in
angiogenesis in vivo.30
Thus, CD31 may play a
role in the invasion, migration, and/or extensions of pseudopodia
through the collagen matrix.
Our results differ from an earlier report by Bach et al,19 which suggested that VE-cadherin, but not CD31, played a role in tube formation in collagen and fibrin gels. However, the model used in the study by Bach and co-workers employed the sandwich methodology as discussed in the introduction. In the sandwich model, endothelial junctions are already formed before addition of the angiogenic stimulus. In contrast, in the three-dimensional suspension model used in the present study, endothelial cells must find each other and link up to form tubes and lumen structures. Thus, CD31/CD31 interactions might be required for the initial formation but not continued maintenance of endothelial cell-cell associations. Consistent with this hypothesis are the observations by Albelda et al31 and Fawcett et al,32 which demonstrated that endothelial cells seeded to culture dishes and grown in the presence of CD31 antibodies fail to establish tight cell contacts whereas addition of the CD31 antibodies to intact monolayers did not inhibit cell-cell contacts or disrupt the cobblestone appearance. Importantly, antibodies directed against CD31 block cytokine-induced corneal neovascularization, indicating that inhibitory effects in the three-dimensional collagen gel model may be predictive of in vivo activity.33
In the presence of VE-cadherin antibodies individual endothelial cells were able to elongate. However, the two VE-cadherin antibodies used resulted in different morphologies, although both morphologies were consistent with an effect on vacuole formation. Cells incubated with the VE-cadherin antibody TEA 1/31 were able to lengthen but remained isolated from adjacent cells. Additionally, nearly one-half of the cells examined did not form visible vacuoles. In contrast, cells incubated the VE-cadherin antibody RM67 also elongated (although shorter than the otherwise untreated PMA plus bFGF plus VEGF group) but contained large vacuoles that apparently had not fused. Additionally, there were also examples of aggregates of three of more cells that exhibited closely apposed, but not fused, vacuoles. These observations support the possibility that the two monoclonal antibodies to VE-cadherin recognize different epitopes (epitope information was not available from the manufacturer) and further suggest that different extracellular domains of VE-cadherin might have different functions in the molecular events involved in tube formation in vitro. Endothelial vacuolization is thought to occur via pinocytosis.18 Consistent with this hypothesis was our observation that addition of free 6-carboxyfluorescein, but not the acetoxymethyl ester, resulted in dye accumulation in the intracellular vacuoles. It is possible that VE-cadherin is involved in some manner in the process of vacuole fusion to form large vacuoles. Cadherins have been previously reported to occur in endocytic membrane vesicles of bovine endothelial cells undergoing junctional remodeling.34 To date, there are no reports of in vivo efficacy (or lack thereof) of anti-VE-cadherin antibodies on angiogenesis.
The mechanism of vascular lumen formation is not well defined, and currently there are two different concepts about how vascular lumens form, and one, or both of them, may be correct. Lumens could form by the coordinated migration of several endothelial cells that maintain their polarity and junctional contacts. However, the idea that intracellular vacuolization may generate lumens dates back to early studies by several groups35,36 that described angiogenic endothelial cells as highly vacuolated. Thus, previous intracellular vacuolization of the endothelial cytoplasm of adjacent endothelial cells could lead to the generation of an intercellular canal by connection of the vacuoles. More recent studies clearly demonstrate a role for VEGF in the formation of vesicular vacuolar organelles,37,38 suggesting that vacuole formation may be a common feature of VEGF-dependent angiogenesis.
In summary, the present study has further characterized a useful new model of angiogenesis. In this model, endothelial cells undergo tube formation and lumen formation over a short time frame. This process is dependent on new mRNA and protein synthesis, thus offering the opportunity to explore, in a coordinated and time-dependent manner, the molecular events that lead to this differentiation.
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Acknowledgements |
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Footnotes |
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S. Yang and J. Graham contributed equally to this work.
Accepted for publication June 18, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is required for kidney tubulogenesis in matrigel cultures in serum free medium. Proc Natl Acad Sci USA 1990, 87:4002-4006vß3 integrin involved in adhesion of leukocytes to endothelium. J Cell Biol 1995, 130:451-460vß3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994, 79:1157-1164[Medline]
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