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From the Departments of Cardiovascular Research,*
Molecular Oncology,
and Pharmacological
Sciences,
Genentech, 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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Hepatocyte growth factor (HGF) is a potent mitogen for a number of cell types, including hepatocytes, myeloid precursor cells, and various epithelial and endothelial cells.11 HGF also promotes epithelial and endothelial cell motility and branching morphogenesis and/or tubular morphogenesis.12,13 HGF may also mediate mesenchymal-epithelial and endothelial interactions, which contribute to tissue repair and embryogenesis.14-16 In vivo, intravenous administration of HGF has been reported to improve collateral formation in ischemic hind limb.17,18 HGF and its receptor c-met, are up-regulated in a number of tumors including breast cancer, urothelial-bladder cancer and gliomas, and like VEGF, HGF levels have been shown to correlate with tumor vascularity.11 HGF can also induce VEGF production by a variety of cells and tissues.17,19,20 In the present study we investigated the possibility that HGF may augment VEGF driven endothelial angiogenesis, independent of the ability to increase VEGF production. To evaluate the in vitro interactions of HGF and VEGF in human umbilical vein endothelial cell (HUVEC) tubulogenesis, we used a modification of type I collagen gel model that incorporates endothelial cells into the collagen before gelation.8 The in vivo activity of HGF and VEGF was evaluated using the rat cornea as previously described.21 We report here that HGF acts as a co-activator (with VEGF) promoting both endothelial survival and tubulogenesis in 3-D collagen gels in vitro and angiogenesis in vivo in the rat cornea. These observations suggest that combination therapy using HGF and VEGF co-administration may be a more effective means to achieve therapeutic angiogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HUVECs were obtained from Clonetics (San Diego, CA) and grown in Clonetics Endothelial Growth Medium (EGM) medium supplemented with 10% fetal bovine serum and endothelial cell growth supplements provided by the manufacturer. Cells from passages 4 to 7 were used throughout the study. Collagen gels (3-D) containing HUVECs were prepared as described previously.8,21 After gelation at 37°C for 30 minutes, the gels were overlaid with 1x basal medium (BM)8 supplemented with phosphate-buffered saline (PBS) (control), HGF, VEGF or the combination of HGF and VEGF at the indicated concentrations. Tube formation was quantified by measuring total tube length (long axis) in three random fields/well at a fixed layer at low-power (x10) magnification using Openlab software (Improvision Inc, Coventry, UK). For each experiment, each group consisted of two to three replicate wells, and all experiments were repeated at least three times. Digital images were captured using Hoffman modulation optics and a Hamamatsu 9600 charge-coupled device camera.
Fibroblast and HUVEC Co-Culture
Neonatal dermal fibroblasts (NDFBs) and FGM2 culture media were purchased from Clonetics (San Diego, CA) and cells were grown in FGM2 supplemented with 10% fetal bovine serum. All cells were used at passages 4 to 6. To co-culture NDFBs with HUVECs, equal ratios of HUVECs and NDFBs (3 x 106 cells/ml each) were mixed and embedded in 3-D collagen gels in BM supplemented with different growth factors as indicated in Results. To distinguish the origin of the tube structures in co-culture experiments, confluent HUVECs grown in monolayer were labeled with Di-I-Ac-LDL (Biomedical Technologies Inc., Stoughton, MA) at final concentration of 10 µg/ml for 4 hours. Cells were then thoroughly washed with PBS, trypsinized, and used as the source of endothelial cells for the co-culture studies. To quantitate the effects of NDFBs on HUVEC tube structure formation, average tube length was measured as described above except that rhodamine fluorescent optics were used in conjunction with the Hoffman modulation optics to confirm that the tubes measured were comprised of endothelial cells. NDFB-conditioned media (CM), was harvested from subconfluent NDFB monolayers that had been incubated in BM for 48 hours. CM were spun at 800 rpm for 5 minutes to remove cell debris and used directly as the culture media for HUVECs in 3-D collagen gels. Controls used media that had not been previously conditioned by the fibroblasts. The HGF content in NDFBs and NDFB-HUVEC co-cultures CM was measured by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Neutralizing antibodies to HGF were purchased from R&D Systems, and the HGF mutants, NK1 and NK2, were provided by Dr. Ralph Schwall (Genentech).
Cell Viability
The collagen gel matrix made conventional methods (eg, trypan blue dye exclusion, annexin V binding) to assess cell viability problematic. Therefore, we assessed cell viability based on nuclear morphology. Gels were fixed in 3.7% buffered formalin and cell nuclei stained with 10 µmol/L 4,6'-diamino-2-phenylindole dihydrochloride (DAPI) for 20 minutes. The cells/tubes were viewed under UV optics to evaluate nuclei. Cells with condensed nuclei or pyknotic nuclei were counted as dead, as were cell ghosts in which nuclei were no longer visibly stained with DAPI. The percentage of viable cells/high-power field was then determined. For each group, six random fields in nine replicate wells were evaluated. In other experiments nuclei were stained with acridine orange and images captured using a Leica confocal microscope.
Real Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) (Taqman) Assay
Total RNA was extracted from HUVECs cultured in 3-D type I
collagen gels for various times in 1x BM supplemented with the
indicated growth factors. A gene-specific PCR oligonucleotide primer
pair and an oligonucleotide probe labeled with a reporter fluorescent
dye at the 5'-end and a quencher fluorescent dye at the 3'-end were
designed using the Oligo 4.0 software (National Bioscience, Plymouth,
MN) following guidelines suggested in the Taqman Model 7700 Sequence
Detection instrument manual (PE Applied Biosystems, Foster City, CA).
The primers and probes used are provided in Table 1
, and mRNA was quantified as described
previously.21
Standard curves for the expression of each
gene were generated by serial dilution of a standard preparation of
total RNA isolated from quiescent HUVECs (ie, maintained 24 hours in
EGM 100% confluency culture media with 10% fetal bovine serum without
additional growth factors) grown in monolayer culture. The mRNA levels
are expressed as the ratio to the housekeeping gene,
glyceraldehyde-3-phosphate dehydrogenase.
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HUVECs grown in 3-D collagen gels for 48 hours were fixed in 1/2 Karnovsky’s solution (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.4) and then washed twice in 0.1 mol/L sodium cacodylate, pH 7.4, (15 minutes per wash) before being postfixed in 1% aqueous osmium tetroxide for 2 hours at room temperature. Following washing in ddH2O the samples were dehydrated through an ethanol series: 50%, 70%, 90%, for 15 minutes each, and then 100% for 2 x 15 minutes, followed by propylene oxide for 2 x 15 minutes. The samples were infiltrated with Eponate 12 (Ted Pella, Redding, CA) first with 1:1 propylene oxide:Eponate 12 overnight, followed by 100% Eponate 12 for 8 hours. The samples were transferred to fresh resin and polymerized in a 60°C oven overnight and ultrathin (80 nm) sections were cut. The sections were stained with uranyl acetate and lead citrate and were observed on a Philips CM12 transmission electron microscope. Images were captured with a GATAN Retractable MultiScan Camera using Digital Micrograph software.
In Vivo Angiogenesis Corneal Assay
In vivo angiogenic activity of the combination of HGF and VEGF was examined by using the rat corneal angiogenesis assay as described previously.21 Hydron pellets containing excipient (control), HGF (50 or 200 ng), VEGF (50 or 200 ng), or the combination of HGF and VEGF (50 or 200 ng each) were implanted into the corneas of 250 to 300 g male Sprague-Dawley rats. All hydron pellets contained 100 ng of sucralfate. At day 6, the animals were euthanized and injected with fluorescein isothiocyanate-dextran to allow for visualization of the vasculature. Corneal whole mounts were made of the enucleated eyes and analyzed for neovascular area using a computer-assisted image analysis (Image Pro-Plus 2.0, Silver Spring, MD).
Statistics
Statistical analyses were performed using one-analysis of variance (INSTAT, GraphPad Software, Sorrento Valley, CA). Multiple comparisons against the control were first analyzed by one-way analysis of variance followed by Bonferroni’s modified Student’s t-test to determine differences between groups. A P value <0.05 was accepted as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When grown in 3-D collagen gels in BM alone, HUVECs underwent
rapid cell death, visible as early as 4 hours and at 24 hours. At 72
hours virtually all of the cells were dead (Figure 1A)
in agreement with earlier
reports.8
Addition of VEGF to the BM, even at
concentrations as high as 800 ng/ml, did not promote cell survival, nor
induce tubulogenesis (Figure 1B)
. HUVECs cultured in 3-D collagen gels
in BM supplemented with 0.4 to 800 ng/ml HGF also underwent rapid
cell death (data not shown except for 200 ng/ml, Figure 1C
).
HGF-treated cells did start to form small vacuoles at 4 hours, but some
of the cells exhibited pyknotic nuclei. At 24 hours, small sprouts were
observed in some cells but numerous dead cells were also observed. Few
viable HUVECs were observed at 72 hours. However, HGF (200 ng/ml), when
combined with VEGF (200 ng/ml), supported cell viability and
tubulogenesis (Figure 1D)
. The sequential changes in cell morphology
were similar to those described in our earlier report detailing
endothelial tubulogenesis in 3-D collagen gels when incubated with
phorbol myristate acetate (PMA) in combination with VEGF and
bFGF.8
Frequent small vacuole-like structures were
observed at 
6 to 8 hours, and an interconnected network of
endothelial cells with lumens was observed by 48 to 72 hours (Figure 1D)
. Figure 2; A, B, and C
, documents the
appearance of the endothelial cells at 4, 24, and 48 hours in 3-D
collagen gels in BM supplemented with HGF and VEGF (200 ng/ml each),
and Figure 2D
shows a representative transmission electron micrograph
of a representative tubular structure demonstrating a lumen-like
structure formed by several adjoining endothelial cells. The
combinatorial effects of HGF with VEGF were both HGF and VEGF
dose-dependent. As shown in Figure 3
,
when the VEGF concentration was fixed at 400 ng/ml, HGF stimulated a
dose-dependent increase in tube formation, with significant effects
observed at 40 and 400 ng/ml HGF. Similar data were obtained when the
HGF concentration was fixed at 400 ng/ml and the VEGF concentration
varied from 0.4 to 400 ng/ml (not shown). These results demonstrated a
synergistic interaction between the two endothelial mitogens, HGF and
VEGF, in the promotion of endothelial survival and tubulogenesis
in vitro.
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VEGF has been reported to be a survival factor for HUVECs when
grown in monolayer in low or no serum.22
Similarly, HGF is
a survival factor for endothelial cells.23
Endothelial
cells were incubated in BM alone or BM supplemented with HGF (200
ng/ml), VEGF (200 ng/ml), or the combination of HGF and VEGF (both at
200 ng/ml), and cell viability (based on nuclear morphology) at 24
hours (confocal microscopy) and 48 hours (DAPI stained nuclei,
examination of multiple high-powered fields) determined as described in
Materials and Methods. As shown in Figure 4
, the majority of cells incubated in BM
alone, VEGF alone, or HGF alone exhibited an apoptotic appearance or
were dead. Representative photomicrographs of pyknotic nuclei and
normal nuclei are shown in Figure 4, B and C
, respectively. However,
the combination of VEGF and HGF greatly improved cell survival, with
80% of the cells in the gel exhibiting normal nuclear morphology at
48 hours. In addition, the cells in the VEGF plus HGF-treated groups
demonstrated interconnected branching networks, as shown above.
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. These results suggest that the survival activity of HGF in
combination with VEGF in 3-D collagen gels might be due in part to the
synergistic induction of the anti-apoptotic genes, A1 and
Bcl-2. HGF Is the Mediator of Endothelial Tubulogenesis in Fibroblast/HUVEC Co-Cultures
Fibroblast and fibroblast-derived factors have been previously
reported to facilitate endothelial morphogenesis into capillary-like
tubes25-27
although the identity of the
fibroblast-derived factor has not been conclusively determined. Because
a major cellular source of HGF is the fibroblast11
we
examined the potential role of HGF as the mediator of
fibroblast-supported endothelial morphogenesis in 3-D collagen gels.
NDFBs were cultured alone and in combination with HUVECs as described.
NDFBs, cultured in BM with VEGF (at concentrations ranging from 0 to
400 ng/ml) were viable and were observed as single, elongated cells
(not shown except at 40 ng/ml VEGF, Figure 5b
). When NDFBs were co-cultured with
HUVECs in BM alone, at least half of the cells were dead, and the
viable cells exhibited a similar morphology to the NDFBs when cultured
without HUVECs (not shown). To determine which cells were viable, the
HUVECs were preloaded with Di-I-Ac-LDL (an endothelial cell-specific
marker) and the co-cultures observed under rhodamine fluorescent
optics. These studies demonstrated that the viable cells (which did not
take up the Di-I-Ac-LDL) were NDFBs (not shown). However, when NDFBs
were co-cultured with HUVECs in BM supplemented with VEGF (40 ng/ml)
elongated network-like structures were observed (Figure 5c)
.
HUVECs preloaded with Di-I-Ac-LDL clearly demonstrated that the
networks were endothelial in origin (Figure 5, d and e)
. CMs were
collected from NDFBs as described in Materials and Methods,
supplemented with VEGF (200 ng/ml), and added to HUVECs in 3-D collagen
gels immediately after gelation. Unconditioned media supplemented with
VEGF (200 ng/ml) were used as the control. HUVECs cultured in NDFB-CM
formed tube-like structures with a time course and morphology
essentially identical to HUVECs cultured in BM supplemented with HGF
and VEGF, although average tube length tended to be reduced (see Figure 6A
). NDFB-CM without VEGF and
unconditioned media with VEGF did not support HUVEC survival or tube
formation in 3-D collagen gels (not shown). The physicochemical
properties of the factor in NDFB-CM indicated the factor was >30 kd,
bound to heparan Sepharose, and was sensitive to heat-inactivation
(90°C, 10 minutes), properties shared by many growth factors
including HGF.
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. Similar data were obtained in NDFB/HUVEC
co-culture experiments (not shown). Two naturally occurring HGF mutants
and antagonists, NK1 and NK2,28
also significantly
inhibited HUVEC tubulogenesis in the co-culture model (Figure 6B)
. We
determined the levels of HGF in the media of the NDFB-HUVEC 3-D gel
co-cultures. At 72 hours, there was 1 ng/ml HGF in the supernatants
recovered from co-cultures in the absence of VEGF, and 750 pg/ml in the
supernatants recovered from the co-cultures when 200 ng/ml VEGF was
added to the BM at time 0. There was no detectable HGF in unconditioned
media. Although the recoverable levels of HGF measured in CM were lower
than those apparently required to promote tube formation, the amount
recovered in the supernatant probably underestimates the local
concentration of HGF in the gels because HGF is known to interact
strongly with heparan-sulfate proteoglycans. Additionally, a
significant amount of HGF may also be bound to cell surface receptors.
Further, the addition of exogenous HGF to the gels at a concentration
of 200 ng/ml may not result in a local concentration of 200 ng/ml.
Based on the antibody neutralization data and antagonism with NK1 and
NK2, the fibroblast facilitation of endothelial tube formation in
co-cultures (with VEGF) is most likely mediated by fibroblast secretion
of HGF. HGF Amplifies VEGF-Driven Angiogenesis in Vivo
Hydron pellets containing excipient (control), HGF, VEGF, or
the combination of HGF and VEGF were implanted into the corneas of
Sprague-Dawley rats. Summary data from this experiment show that
pellets containing the combination of VEGF and HGF demonstrated a
significant increase in vessel area compared with either growth factor
alone (Figure 7, A–E)
. Results from this
experiment were repeated in two separate experiments. These in
vivo data strongly support our in vitro observations
that HGF can augment VEGF-driven angiogenesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HGF shares many properties with bFGF and VEGF. It is a potent endothelial cell mitogen, and it stimulates endothelial migration and invasion through extracellular matrix proteins. However, in contrast to VEGF and bFGF, HGF is a potent morphogen for multiple cell types.11 For example, in addition to promoting endothelial organization into tubule-like structures on Matrigel and on the surface of collagen gels, HGF will also induce Madin-Darby canine kidney epithelial cells to organize into a branching network of tubules with the proper apical-basolateral polarity,35 and mammary epithelial cells to form duct-like structures.28 HGF synergistically interacts with VEGF to induce endothelial cell proliferation and migration (in monolayer cultures).17 We and others8-10 have observed that HUVECs cultured in 3-D collagen gels do not proliferate (independent of the mixture of growth factors used), but undergo differentiation into tubule-like structures. In this 3-D model, we and others8-10 have observed that the combination of bFGF and VEGF did not support cell survival or tubulogenesis. For example, Ilan and co-workers9 demonstrated that HUVECs cultured in 3-D collagen gels (under conditions virtually identical to those used in the present study) in the presence of VEGF (20 ng/ml) underwent rapid apoptosis as indicated by terminal dUTP nick-end labeling staining, nuclear morphology, and poly(ADP-ribose) polymerase hydrolysis. Previous work by Ilan, and others9,10 demonstrated that PMA, alone or in combination with bFGF and VEGF could support cell survival and branching morphogenesis. Because PMA is not a naturally occurring stimulus, in preliminary experiments we tested a number of different growth factors and cytokines in combination with either VEGF or bFGF. As shown in the present study, the combination of HGF and VEGF (or HGF and bFGF, data not shown) enabled endothelial cell survival and morphogenesis into a network of tubule-like structures. These observations suggest that the synergistic interactions between the HGF and VEGF signaling pathways differ from those of bFGF and VEGF co-administration. The synergy between VEGF and HGF was clearly demonstrated by the more than additive changes in the mRNA levels of bcl-2, A1, cell survival indices and tubule formation. Further, the inability of even very high concentrations of VEGF to support endothelial survival or tubulogenesis in the 3-D collagen gel model suggests that the synergistic actions of HGF are likely independent of further increases in VEGF production.
The receptor for HGF, c-met, is expressed on endothelial and
smooth muscle cells as well as pericytes,36
and HGF
stimulates the proliferation of all of these vascular cell types
in vitro.11
Activation of c-met is
known to stimulate phosphatidylinositol-3-kinase, phospholipase C
,
pp60src, and Grb2/Sos1; signaling pathways also
activated by VEGF.37
However, the observed synergistic
interactions between VEGF and HGF suggest that there may be differences
in the signal transduction pathways. For example, induction of
epithelial tubules by HGF is dependent on the STAT
pathway.38
A role of the STAT pathway in VEGF-mediated
responses has not been reported. There are undoubtedly other
differences in the nature of cellular responses to VEGF and HGF that
have yet to be discovered.
Hypoxia is potent inducer of VEGF mRNA and protein, and the expression of the VEGF receptors kdr and flt-1 are also increased in response to ischemia.1 However, HGF expression is reduced in diseased segments of blood vessels from patients with critical limb ischemia compared to disease-free segments.18 HGF mRNA and protein can be down-regulated by hypoxia,39 although this may be a time- and tissue-dependent phenomenon.15,40 In patients as well as in a myocardial ischemia model, c-met is up-regulated in response to hypoxia.40-42 Down-regulation of HGF in response to hypoxia may be an important contributor to the pathology associated with peripheral vascular disease, and the up-regulation of c-met in peripheral vascular disease may offer an opportunity for therapeutic intervention with HGF.
In summary, in the present study we have demonstrated that HGF and VEGF act in synergy to induce human endothelial morphogenesis into tube-like structures in 3-D collagen gels. These morphogenic changes are accompanied by synergistic induction of the anti-apoptotic genes (bcl-2 and A1). Our in vitro and in vivo results demonstrate that by acting in concert with VEGF, HGF may amplify an angiogenic response. These observations highlight the importance of biological context, because the activity of any angiogenesis-regulating cytokine will likely depend on the presence and concentration of other cytokines in the pericellular environment. Angiogenesis is a multistep process orchestrated by a complex mixture of different growth factors, cytokines, proteolytic enzymes, and matrix molecules and thus the optimal therapy to induce a functional angiogenic response may require a mixture of the appropriate factors.
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Acknowledgements |
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Footnotes |
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Accepted for publication November 29, 2000.
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