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From the Cutaneous Biology Research Center, Department ofDermatology,* Massachusetts General Hospital and HarvardMedical School, Charlestown; and the Department ofPathology, Beth Israel Deaconess MedicalCenter and Harvard Medical School, Boston, Massachusetts
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-smooth muscle actin-positive periendothelial cells was
significantly increased, indicative of an increased vascular
maturation status. These findings identify an inhibitory role of
Ang1/Tie2 receptor-mediated vessel maturation in SCC growth and suggest
that up-regulation of its antagonist, Ang2, during
early-stage epithelial tumorigenesis contributes to the angiogenic
switch by counteracting specific vessel-stabilizing effects of
Ang1.
Recently, a novel endothelial-specific receptor tyrosine kinase, Tie2, was identified that has been implicated in vascular maintenance as well as in angiogenesis.7-9 Angiopoietin-1 (Ang1) specifically induces tyrosine phosphorylation of Tie2, whereas Ang2, a second ligand for the Tie2 receptor, has been identified as a naturally occurring antagonist for Ang1, blocking its ability to induce Tie2 tyrosine kinase activity in endothelial cells.10 The angiopoietin/Tie2 system acts in a complementary manner with VEGF and plays an important role in late stages of vascular development.11 Thus, in mouse embryos lacking either Ang1 or Tie2, the early stages of VEGF-dependent vascular development occur rather normally and result in the formation of a primitive vasculature, whereas maturation and stabilization of the primitive vasculature are severely perturbed.12-14 Consistent with its proposed role as an Ang1 antagonist, transgenic overexpression of Ang2 in endothelial cells results in lethal embryonic defects comparable to those observed in Ang1- and Tie2-deficient mice.10 Increasing evidence suggests that the mechanism of action of angiopoietins involves interactions between endothelial cells and pericytes.15 Ang1 engagement of Tie2 has been proposed to stabilize mature blood vessels in the adult vasculature by promoting the interaction between endothelial cells and supporting periendothelial cells. In contrast, Ang2 has been thought to block the constitutive stabilizing effects of Ang1, thereby facilitating the angiogenic response to VEGF, or vessel regression in the absence of VEGF.10,15
There is increasing evidence that the angiopoietin/Tie2 system also plays an important role in tumor angiogenesis; however, the distinct biological effects of Ang1 and Ang2 on tumor growth and angiogenesis have remained controversial16-20 and the regulation of expression of both factors during the consecutive stages of multistep carcinogenesis has remained unknown. In this study, we present evidence for an inhibitory role of Ang1-mediated Tie2 activation on squamous cell carcinoma (SCC) growth. Expression of Ang2, but not of Ang1, was strongly up-regulated in angiogenic tumor vessels during the consecutive stages of mouse skin carcinogenesis. Overexpression of Ang1 in A431 SCCs enhanced Tie2 receptor phosphorylation in vivo and resulted in a significant inhibition of tumor growth whereas overexpression of Ang2 did not modulate Tie2 phosphorylation and accordingly failed to affect tumor growth and angiogenesis. Moreover, Tie2 engagement by Ang1 significantly increased the fraction of mature blood vessels in A431 tumors, suggesting an important role of the maturation status of the tumor vasculature in tumor progression.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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For tumor initiation, 25 µg of 7,12-dimethylbenz(d)anthracene (DMBA) (Sigma, St. Louis, MO) dissolved in 200 µl of acetone, were topically applied to the shaved back skin of 8-week-old female FVB mice, followed by weekly topical application of 5 µg of the tumor promoter PMA (Sigma) throughout 20 weeks as described.21 Skin and tumor samples were embedded in OCT compound and frozen in liquid nitrogen for cryostat sectioning.
In Situ Hybridization
In situ hybridization was performed on 6-µm-thick cryostat sections with sense and anti-sense single-stranded 35S-labeled RNA probes for Ang1 and Ang2 as described.22
Cell Culture
The human SCC cell line A431 was obtained from the American Type Culture Collection (Rockville, MD) and was maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc., Grand Island, NY) containing 10% fetal bovine serum, 4.5 mg/ml glucose, 2 mmol/L L-glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies, Inc.). Human dermal microvascular endothelial cells (HDMECs) were isolated from neonatal human foreskins and were cultivated as described.23
Cell Transfection
A 2.0-kb full-length human Ang1 cDNA24 and a 2.3-kb full-length human Ang2 cDNA,10 were kindly provided by Dr. George D. Yancopoulos (Regeneron Pharmaceuticals Inc., Tarrytown, NY). Vectors for stable expression of human Ang1 and Ang2 were generated by subcloning of a 2.0-kb Ang1 cDNA and of a 2.3-kb Ang2 cDNA, comprising the complete coding sequences, into the XhoI (Ang1) or EcoRI (Ang2) sites of the pCDNA3.1Zeo(-) expression vector (Invitrogen, San Diego, CA) that contains a CMV-enhancer-promoter and a Zeocin selection cassette. The sequences and correct orientation were verified by restriction mapping and by direct sequencing using the Sanger dideoxy method. A431 cells were stably transfected with pCDNA/Ang1, pCDNA/Ang2, or pCDNA vector control, respectively, by using the SuperFect transfection reagent (Qiagen, Chatsworth, CA) and 10 µg of each plasmid according to the manufacturer’s recommendations. Stably transfected clones were selected in medium containing 250 µg/ml of Zeocin (Invitrogen) for 3 to 4 weeks, and 35 clones for each construct were isolated and analyzed for Ang1 and Ang2 expression.
Cell Growth and Apoptosis Assays
To analyze anchorage-dependent cell growth, 5 x 104 A431 cells were plated in duplicate into 100-mm culture dishes, and the total cell numbers per dish were determined after 1 day (N0) and after 7 days (N) at the end of the growth period (t) by using a hemocytometer. The cell-doubling time (T) was calculated by using the following formula: T = ln2 x t/lnN/N0. Anchorage-independent cell growth was measured using a soft agar assay as described.25 Briefly, 1 x 104 control-transfected or parental A431 cells, or cells transfected with Ang1 or Ang2 were transferred into six 35-mm cell-culture dishes with 2-mm grids (Nunc, Naperville, IL), and colonies were counted after 8 days. Apoptosis induced by serum withdrawal was studied in subconfluent A431 cell clones after 6 days of culture in serum-free medium. The percentage of apoptotic cells was determined as described,26 using the Fluorescein-FragEL DNA fragmentation kit (Oncogene, Cambridge, MA) according to the manufacturer’s instructions, and a Becton-Dickinson (Franklin Lanes, NJ) FACS-Scan.
Northern Blot Analysis
Total cellular RNA was isolated from confluent cell cultures and tumor tissues using the RNeasy kit (Qiagen). Ten µg of RNA were fractionated by electrophoresis on 1% agarose formaldehyde gels and were transferred to Biotrans nylon-supported membranes (ICN Pharmaceuticals, Costa Mesa, CA) as described.27 32P-radiolabeled cDNA probes were prepared with a random primed synthesis kit (Multiprime; Amersham, Arlington Heights, IL). mRNAs for Ang1, Ang2, and VEGF were detected with a 2.0-kbp human Ang1 cDNA, a 2.3-kbp human Ang2 cDNA, or a 204-bp human VEGF cDNA that recognizes all known VEGF variants.28 A human 36B4 cDNA probe29 was used as a control for equal RNA loading. Blots were washed at high stringency and exposed to X-OMAT MR film (Kodak, Rochester, NY) for varying times.
Receptor Phosphorylation Assays
To analyze the biological activity of transfected Ang1 and Ang2, an in vitro Tie2 receptor phosphorylation assay was used. Conditioned media (CM) were obtained from confluent stably transfected A431 cells grown for 48 hours in serum-free medium and were concentrated 50-fold by ultrafiltration (10,000 MWCO; Amicon, Inc., Beverly, MA). Protein concentrations were determined using the BioRad protein assay (BioRad, Hercules, CA). HDMECs grown to 80% confluence and serum-starved overnight were left unchallenged or were challenged for 10 minutes with recombinant Ang1* (150 ng/ml), recombinant Ang1*10 (150 ng/ml) plus recombinant Ang2 (rAng-2, 500 ng/ml), with CM obtained from Ang1 or vector control transfectants, or with recombinant Ang1* plus CM harvested from Ang2 transfectants, respectively. Recombinant Ang1* and recombinant Ang210 were generously provided by Dr. George D. Yancopoulos. Cells were solubilized in lysis buffer (1% Triton X-100, 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 100 mmol/L sodium fluoride, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin, and 10 µg/ml of aprotinin), and Tie2 was immunoprecipitated from the soluble fraction using a polyclonal antibody reactive with the carboxyl terminus of Tie2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4°C overnight. After incubation with protein A-Sepharose (Amersham Pharmacia Biotech, Inc., Piscataway, NJ), immunocomplexes were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Bio-Rad). Tie2 phosphorylation was analyzed by probing the membranes with a monoclonal anti-phosphotyrosine antibody (PY-20; ICN Biomedicals, Inc., Aurora, OH). The same membranes were then stripped using TBST (10 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.1% Tween 20, pH 2.4) overnight at room temperature and were reprobed for Tie2 with a rabbit anti-Tie2 antibody (Santa Cruz Biotechnology, Inc.). In vivo receptor phosphorylation assays were performed as recently described.30 Briefly, samples obtained from three different vector control-transfected and Ang1- or Ang2-transfected A431 tumors were pooled and homogenized in RIPA lysis buffer containing protease inhibitors as above. Protein samples were immunoprecipitated using antibodies against Tie2 (RG133; kindly provided by Dr. Peter Maisonpierre, Regeneron Pharmaceuticals, Inc.) or mouse VEGFR-2 (flk-1; Santa Cruz Biotechnology, Inc.). Immunocomplexes were recovered on protein G-Sepharose and separated by 7.5% SDS-PAGE, transferred onto blotting membrane, and then probed with antibodies against phosphotyrosine (4G10; Upstate Biotechnology, Inc., Lake Placid, NY), Tie2 (Santa Cruz Biotechnology, Inc.), or VEGFR-2 (Santa Cruz Biotechnology, Inc.). Immunoreactive proteins were visualized using a chemiluminescence detection system (ECL; Amersham). Levels of Tie2 phosphorylation were quantified by densitometry and were normalized to the expression levels of Tie2.
Reverse Transcriptase-Linked Polymerase Chain Reaction (RT-PCR)
RT-PCR of Tie1 and Tie2 mRNA expression in A431 clones was performed using 0.2 µg of total RNA as a template and reagents supplied by the GeneAmp RNA PCR Kit (Applied Biosystems, Foster City, CA). For PCR amplification, the following primer sequences were selected from different exons of the human Tie1 and Tie2 genes: Tie1, 5'-TTTAACCCTGGTGTGCATCC-3' (sense) and 5'-CCGCAGAAAATCTAGCAGGT-3' (anti-sense; 451-bp PCR product); Tie2,5'-AGTTCGAGGAGAGGCAATCA-3' (sense) and 5'-CCGAGGTGAAGAGGTTTCCT-3' (anti-sense; 290-bp PCR product). The primer sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Stratagene, La Jolla, CA) were 5'-CCACCCATGG CAAATTCCATGGCA-3' (sense) and 5'-TCTAGACGGCAGGTCAGGTCCACC-3' (anti-sense; 600-bp PCR product). HDMECs at passage 6 were used as a positive control. Cycling parameters were as follows: Tie1 and Tie2: denaturation, 94°C for 30 seconds; annealing, 53°C for 30 seconds; extension, 65°C for 30 seconds, with 35 cycles. GAPDH: denaturation, 94°C for 45 seconds; annealing 60°C for 45 seconds; extension, 72°C for 90 seconds, with 35 cycles. PCR products were electrophoresed in 3% agarose gels and were visualized by ethidium bromide staining. For analysis of Ang1 and Ang2 mRNA expression in tumors real-time RT-PCR was performed using 100 ng of RQ-DNase-treated total RNA per each reaction. The sequences of the primer/dual labeled probe sets were as follows: Ang1: 5'-TGTTAACAGGAGGATGGTGGTTT-3' (sense),FAM-TGCTTGTGGCCCCTCCAATCTAAATG-TAMRA (dual-labeled probe), and5'-GTTTTGTCCCGCAGTATAGAACATT-3' (anti-sense). Ang2: 5'-GCCGCTCGAATACGATGACT-3', FAM-TGCAGAGGCTGCAAGTGCTGGAGAA-TAMRA (dual-labeled probe), and 5'-CCACTGAGTGTTGTTTT CCATGA-3' (anti-sense). Each reaction was performed in the presence of a GAPDH internal control detection system using the Taqman EZ RT-PCR Core Reagent (Applied Biosystems). All reactions were performed in triplicates using the ABI Sequence Detection System 7000 (Applied Biosystems).
Tumorigenicity Assay
Stably transfected A431 cells (2 x 106
in 100 µl of serum-free culture medium) were injected intradermally
into both flanks of 8-week-old female Swiss (nu/nu) mice (five mice for
each clone). Two vector-transfected clones, three Ang1- or
Ang2-overexpressing clones, and the parental cell line were
studied. The smallest and largest tumor diameter were measured weekly
using a digital caliper, and tumor volumes were calculated using the
following formula: volume = 4/3 x 
x (1/2 x
smaller diameter)2
x 1/2 x larger
diameter. Tumor data were analyzed using the two-sided unpaired
t-test. Tumors were harvested after 3 weeks and were either
embedded in OCT compound and frozen in liquid nitrogen for cryostat
sectioning, or were fixed in formalin and embedded in paraffin for
routine histology. For RNA extractions, tumors were snap-frozen in
liquid nitrogen. In an additional experiment one vector-transfected
clone, one Ang1-overexpressing clone, and one Ang2-overexpressing clone
(2 x 106
cells/injection) or a 1:1 mixture
of one vector control and one Ang1-transfected clone or a mixture of
one Ang1-transfected and one Ang2-transfected clone (1 x
106
cells each clone/injection) were injected
(n = 10 tumors per group). All animal studies
were approved by the Massachusetts General Hospital Subcommittee on
Research Animal Care.
Western Blot Analysis
Tumors were snap-frozen in liquid nitrogen and homogenized in lysis buffer (2% SDS, 50 mmol/L Tris, pH 7.4, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin, and 10 µg/ml of aprotinin). Fifty µg of protein per sample were analyzed by denaturing SDS/PAGE and immunoblotted with a rabbit antibody against human VEGF (kindly provided by Dr. Donald R. Senger, Harvard Medical School, Boston, MA).
Computer-Assisted Morphometric Analysis of Tumor Vessels
Cryostat sections (6 µm) were stained with a rat anti-mouse platelet-endothelial cell adhesion molecule-1 (CD31) monoclonal antibody (Pharmingen, San Diego, CA) as previously described.31 Representative sections obtained from five A431 tumors from each cell clone were analyzed using a Nikon E-600 microscope (Melville, NY). Images were captured with a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI), and morphometric analyses were performed using the IP LAB software (Scanalytics, Billerica, MA) as described.32 Three different fields in each section were examined at x10 magnification, and the number of vessels per mm2, the vessel size, and the relative area occupied by tumor blood vessels within the tumors were determined. The two-sided unpaired t-test was used to analyze differences in microvessel density, vessel size, and total vascular area.
Indirect Immunofluorescence
Cryosections were fixed and stained as previously
described,4
using monoclonal antibodies against mouse CD31
(dilution, 1:50; Pharmingen), or against -smooth muscle actin
(1:100, -SMA, Cl 14A; DAKO, Glostrup, Denmark), expressed by
vascular smooth muscle cells and pericytes. The secondary antibodies,
labeled with either Texas Red or fluorescein isothiocyanate (Jackson
ImmunoResearch, West Grove, PA) were used at a 1:50 dilution. Sections
were incubated, together with the secondary antibody, with 20 µg/ml
of Hoechst bisbenzimide (Sigma Chemical Co.) for staining of cell
nuclei. Specimens were mounted in Mowiol (Calbiochem, La Jolla, CA),
and conventional fluorescent and light microscopy were performed on a
Nikon E-600 microscope. Three independent fields per section obtained
from each cell clone were analyzed, and the percentage of blood vessels
associated with -SMA-positive cells was defined as the vessel
maturation index.33
The two-sided unpaired
t-test was used to analyze differences in the vessel
maturation index.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We first investigated the regulation of Ang1 and Ang2 mRNA
expression during chemically induced two-step mouse skin carcinogenesis
by in situ hybridization. In normal skin, Ang1 was
constitutively expressed at low levels by mesenchymal stroma cells in
the vascularized dermis (Figure 1, A and D)
, whereas no Ang2 mRNA was detectable (Figure 1, G and J)
. The
expression of Ang2 (Figure 1
; G to L), but not of Ang1 (Figure 1
; A to
F), was strongly up-regulated throughout the consecutive stages of
mouse skin carcinogenesis. Enhanced Ang2 expression signals were
detected in endothelial cells of tumor-associated blood vessels in
benign papillomas (Figure 1, H and K)
and even more pronounced in SCC
(Figure 1, I and L)
. Ang1 or Ang2 were not expressed by tumor cells in
benign papillomas (Figure 1
; B, E, H, and K) or in SCCs (Figure 1
; C,
F, I, and L). These results identified up-regulation of Ang2 mRNA
expression as an early event during multistep carcinogenesis. We next
investigated whether the switch to Ang2 expression also occurred in an
established mouse model of SCC using intradermally transplanted human
A431 SCC. A431 tumor cells did not express Ang1 (Figure 2, A and C)
or Ang2 (Figure 2, B and D)
.
Little Ang1 expression was detectable in mesenchymal stroma cells
(Figure 2
; A, C, and E), whereas Ang2 mRNA was strongly expressed by
endothelial cells of tumor blood vessels (Figure 2
; B, D, and F).
Control hybridizations with sense riboprobes for both Ang1 (Figure 2, G and I)
and Ang2 (Figure 2, H and J)
revealed very low background
signals. Taken together, these findings suggested that enhanced Ang2
expression might contribute to tumor progression and angiogenesis by
blocking the constitutive stabilizing effects of Ang1.
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To investigate the distinct effects of Ang1 versus Ang2
on tumor growth and angiogenesis, A431 cells were stably transfected to
constitutively overexpress Ang1 or Ang2. As determined by Northern
analysis, parental A431 cells and vector-transfected control clones
did not express any detectable amounts of Ang1 or Ang2 mRNA
in vitro (Figure 3A)
,
whereas three different Ang1- and Ang2-transfected clones strongly
expressed the mRNA for Ang1 or Ang2, respectively (Figure 3A)
. We
next performed Tie2 receptor phosphorylation assays in HDMECs to
analyze the biological activity of transfected Ang1 and Ang2.
Recombinant Ang1* (150 ng/ml) induced tyrosine phosphorylation of Tie2.
This induction was blocked by addition of recombinant Ang2 (500 ng/ml),
confirming the functional activity of the assay (Figure 3B)
. CM
obtained from Ang1-transfected A431 clones potently induced tyrosine
phosphorylation of Tie2, as compared with CM obtained from A431 cells
transfected with vector only (Figure 3B)
. In contrast, CM harvested
from Ang2-overexpressing A431 clones potently blocked Ang1*-mediated
Tie2 phosphorylation (Figure 3B)
. These findings confirmed the
biological activity of Ang1 and Ang2 secreted by stable transfectants.
They are in agreement with the proposed role of Ang1 acting as an
agonist for the Tie2 receptor and of Ang2 acting as an Ang1 antagonist
at the level of the Tie2 receptor.
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Several in vitro assays were performed to detect
potential phenotypic differences between control and Ang1- and
Ang2-transfected A431 SCC clones. Anchorage-independent growth was
determined to evaluate the maintenance of the transformed phenotype and
was studied by determination of colony numbers in a soft agar assay. No
significant differences in the number of colonies were observed between
the different transfected A431 clones and parental A431 cells (Figure 4A)
. In accordance, cell-doubling times
under anchorage-dependent culture conditions in Ang1- and
Ang2-overexpressing clones were comparable to those observed in
vector-transfected or parental A431 control cells (Figure 4B)
. RT-PCR
analyses (Figure 4C)
demonstrated the complete absence of Tie2 and
orphan Tie1 receptor mRNA expression in vector-control and Ang1- and
Ang2-transfected A431 cells, whereas expression of both Tie2 and Tie1
mRNA was clearly detected in cultured HDMECs used as positive control.
To compare the susceptibility to induction of apoptosis, A431 clones
were cultured under serum-free conditions for 6 days. No significant
differences in the percentage of apoptotic cells were found between
Ang1 (6.8% ± 1.0), Ang2 (8.7% ± 3.0), or control transfected clones
(12.5% ± 6.3). Moreover, no alterations in cellular morphology were
detected (data not shown).
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After intradermal injection into immunodeficient nude mice,
parental A431 cells and vector-transfected control cells formed rapidly
growing SCCs, reaching a volume of 1500 to 1800
mm3
within 3 weeks (Figure 5A)
. In contrast, overexpression of Ang1
resulted in a significant inhibition of tumor growth by more than 70%
(P < 0.001, Figure 5A
). Even at 5 weeks after
tumor injection, Ang1-overexpressing tumors did not reach the size of
control tumors at 3 weeks after injection (data not shown).
Surprisingly, overexpression of Ang2 in A431 tumors had no effect on
tumor growth (Figure 5A)
. Northern blot analysis of RNA extracted from
representative tumors confirmed that Ang1- and Ang2-transfected tumors
maintained their respective mRNA expression in vivo
throughout the entire observation period (data not shown). By using
quantitative real-time RT-PCR we found that the average expression of
endogenous Ang2 in control tumors was 115-fold higher than the average
expression of endogenous Ang1. However, in Ang1-transfected tumors, the
average expression of recombinant Ang1 was more than 74-fold higher
than the expression of endogenous Ang2. In Ang2-transfected tumors, the
average expression of recombinant Ang2 was more than 9 times higher
than the endogenous Ang2 expression. In accordance, in vivo
Tie2 phosphorylation assays demonstrated that overexpression of Ang1
resulted in increase of phosphorylation levels of the Tie2 receptor
in vivo as compared with vector-transfected controls
(2.6-fold induction, Figure 5B
). No major differences in the expression
levels of Tie2 were found between control and Ang1-transfected tumors
(Figure 5B)
. In Ang2-overexpressing tumors, the phosphorylation levels
of Tie2 were comparable to those of control tumors (Figure 5B)
. These
results demonstrated that Ang1-mediated activation of the Tie2 receptor
resulted in inhibition of tumor growth.
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VEGF and the angiopoietins play complementary and coordinated
roles in angiogenesis.11
We next studied whether the
expression of VEGF was modulated by overexpression of Ang1 or Ang2.
In situ hybridizations revealed that VEGF mRNA was expressed
at comparable levels in the different A431 tumors (Figure 6
; A to F). These findings were confirmed
by Northern blot analysis (data not shown). Moreover, no significant
differences of VEGF protein levels were found between control and Ang1-
and Ang2-transfected tumors, as studied by Western blot analysis
(Figure 6G)
. Importantly, immunoprecipitation studies of tumor lysates
revealed similar expression and phosphorylation levels of VEGFR-2
(flk-1) in tumors of all types (Figure 6H)
.
|
We analyzed the tumor-associated vasculature, visualized with an
antibody to CD31. Immunostains showed that blood vessels were
distributed rather uniformly throughout the viable tumor areas in all
experimental groups (Figure 7A)
.
Moreover, morphometric analyses of tumor-associated blood vessels did
not reveal any significant differences of vascular density or average
vessel size (Figure 7, B and C)
between control tumors and Ang1- or
Ang2-overexpressing tumors. Because the mechanism of action of
angiopoietins may involve critical interactions between endothelial
cells and pericytes,11,15
we next investigated the effects
of Ang1 and Ang2 overexpression on the maturation status of the tumor
vasculature, using double-immunofluorescent stains for CD31 (tumor
endothelial cells) and SMA (smooth muscle cells/pericytes).
Ang1-overexpressing tumors exhibited an increased number of blood
vessels surrounded by SMA-positive periendothelial cells, as compared
with control and Ang2-overexpressing tumors (Figure 8
; A to C). The maturation index, defined
as the fraction of blood vessels associated with SMA-positive
periendothelial cells, was significantly increased, by more than 50%,
in Ang1-overexpressing tumors as compared with control tumors
(P < 0.05; Figure 8D
). No major differences
were found between control and Ang2-overexpressing tumors (Figure 8D)
.
These findings demonstrate that overexpression of Ang1 in tumors
results in enhanced ratios of mature blood vessels, possibly by
counteracting the destabilizing effects of endothelium-derived Ang2.
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To investigate whether Ang2 might rescue the reduced growth of
Ang1-overexpressing tumors, we performed additional in vivo
studies with mixtures of Ang1- and Ang2-overexpressing cells. In
accordance with the results above, tumor growth was inhibited by
overexpression of Ang1 whereas overexpression of Ang2 had no effect
(Figure 9)
. The admixture of
Ang2-overexpressing cells to Ang1-overexpressing cells (1:1) greatly
diminished the inhibition of tumor growth mediated by Ang1
(P < 0.001). Admixture of vector
control-transfected cells to Ang1 overexpressing only slightly
diminished the inhibitory effects of Ang1.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Controversial findings have been reported regarding the effects of Ang1 versus Ang2 expression on the growth and angiogenesis of established tumors. Whereas Ang1 inhibited MCF-7 breast cancer growth,16 recent studies in HT29 colon,18 TA3 mammary, and Lewis lung carcinomas17 did not detect major effects of Ang1 overexpression on in vivo tumor growth. In addition, overexpression of Ang2 has been found to either promote18-20 or to inhibit17 in vivo growth of various tumor xenografts. In these experimental tumor studies, the in vivo Tie2 phosphorylation levels in response to Ang1 and Ang2 overexpression had not been assessed.
To study the effects of de novo Ang1 or Ang2 expression on in vivo Tie2 phosphorylation, angiogenesis, and tumor growth, we made use of an established orthotopic xenograft mouse model,25,32 using human A431 SCC cells. The A431 cell line was chosen because of the complete absence of Ang1 and Ang2 expression in these cells and because we found an expression pattern of Ang1 and Ang2 in A431 xenografts that was comparable to that observed during multistep mouse skin carcinogenesis. Our results demonstrate that overexpression of Ang1 promoted the interactions between endothelial cells and periendothelial cells in human SCCs. The observed increase in the percentage of mature blood vessels was consistent with the marked induction of Tie2 phosphorylation in vivo. These data are in agreement with the recently proposed vessel-stabilizing role of Ang1.15 Whereas activated tumor endothelium may promote tumor progression,4,37,38 our results indicate that increased maturation of tumor vessels may result in inhibition of tumor growth because overexpression of Ang1 potently inhibited the growth of A431 SCCs by more than 70%. The potential relevance of these findings to human disease is supported by the recently reported inverse correlation between the expression of Ang1 and the size of human thyroid tumors.39
The exact mechanisms by which Ang1-mediated vessel maturation impairs tumor growth remain to be established. Pericytes and perivascular smooth muscle cells have been shown to play major roles in maintaining vessel maturation and integrity.12-14,40-42 Thus, as a result of the increased fraction of mature tumor vessels that are surrounded by support cells in Ang1-overexpressing tumor xenografts, tumor angiogenesis may be functionally inhibited. VEGF-induced microvascular hyperpermeability represents one of the earliest steps in the angiogenic cascade and provides a matrix that facilitates in-growth of blood vessels43 and tumor progression.38 Because Ang1 inhibits vascular permeability44 it is conceivable that Ang1 overexpression also resulted in a less submissive matrix for tumor growth and progression. Importantly, VEGF mRNA and protein expression in Ang1-overexpressing tumors were comparable to vector control-transfected tumors. Moreover, expression and phosphorylation of VEGFR-2 were also comparable in all tumors studied, suggesting that tumor growth retardation was not mediated by modulation of VEGF activity.
Overexpression of Ang2 did not modulate Tie2 phosphorylation and accordingly failed to affect tumor growth and angiogenesis. It has recently been reported that overexpression of Ang2 in colon and hepatocellular cancer resulted in enhanced tumor growth.18,19 This may be explained by the reported expression of endogenous Ang1 in those tumor cells, in contrast to A431 cells that do not express any detectable levels of Ang1. It is therefore conceivable that in the presence of tumor cell-derived Ang1, overexpression of Ang2 may promote tumor growth by blocking the growth inhibitory Ang1 signal. This is in agreement with our findings that the inhibitory effect of Ang1 on tumor growth could be partially rescued by Ang2. Moreover, our results indicate that the biological effects of Ang1 and Ang2 are critically dependent on their relative expression levels and consequently on the phosphorylation status of the Tie2 receptor. Therefore, correlation of intratumoral angiopoietin expression with Tie2 phosphorylation is essential to assess the distinct roles of Ang1 versus Ang2 expression for tumor growth and angiogenesis. Several lines of evidence strongly indicate that the observed effects of transfected Ang1 and Ang2 on tumor growth were not because of direct effects on tumor cells, because A431 cells do not express the Tie2 receptor and no differences in the growth rate, colony forming ability, or apoptosis rate were observed between the different transfected clones in vitro.
In summary, we have identified the up-regulation of Ang2 expression as an early event during multistep carcinogenesis. Moreover, we provide evidence for an inhibitory role of Ang1-mediated Tie2 receptor activation in SCC growth. Finally, our findings suggest an important function of the maturation status of the tumor vasculature in tumor progression.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health/National Cancer Institute (grants CA69184 and CA 86410 to M. D.), the American Cancer Society Program Project (grant 99-23901 to M. D.), the Deutsche Forschungsgemeinschaft (to T. H. and B. L.-A.), the Human Frontiers Science Program (to M. Sk.), the Dermatology Foundation (to M. St.), and the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement (to M. D.).
Accepted for publication January 10, 2002.
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 A. Tyagi, R. P. Singh, K. Ramasamy, K. Raina, E. F. Redente, L. D. Dwyer-Nield, R. A. Radcliffe, A. M. Malkinson, and R. Agarwal Growth Inhibition and Regression of Lung Tumors by Silibinin: Modulation of Angiogenesis by Macrophage-Associated Cytokines and Nuclear Factor-{kappa}B and Signal Transducers and Activators of Transcription 3 Cancer Prevention Research, January 1, 2009; 2(1): 74 - 83. [Abstract] [Full Text] [PDF]
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 T. Szarvas, T. Jager, M. Totsch, F. Vom Dorp, C. Kempkensteffen, I. Kovalszky, I. Romics, S. Ergun, and H. Rubben Angiogenic Switch of Angiopietins-Tie2 System and Its Prognostic Value in Bladder Cancer Clin. Cancer Res., December 15, 2008; 14(24): 8253 - 8262. [Abstract] [Full Text] [PDF]
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 J. Cai, O. Kehoe, G. M. Smith, P. Hykin, and M. E. Boulton The Angiopoietin/Tie-2 System Regulates Pericyte Survival and Recruitment in Diabetic Retinopathy Invest. Ophthalmol. Vis. Sci., May 1, 2008; 49(5): 2163 - 2171. [Abstract] [Full Text] [PDF]
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 W. S.N. Shim, I. A.W. Ho, and P. E.H. Wong Angiopoietin: A TIE(d) Balance in Tumor Angiogenesis Mol. Cancer Res., July 1, 2007; 5(7): 655 - 665. [Abstract] [Full Text] [PDF]
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 F.-C. Ye, D. J. Blackbourn, M. Mengel, J.-P. Xie, L.-W. Qian, W. Greene, I-T. Yeh, D. Graham, and S.-J. Gao Kaposi's Sarcoma-Associated Herpesvirus Promotes Angiogenesis by Inducing Angiopoietin-2 Expression via AP-1 and Ets1 J. Virol., April 15, 2007; 81(8): 3980 - 3991. [Abstract] [Full Text] [PDF]
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 A. P. Hall Review of the Pericyte during Angiogenesis and its Role in Cancer and Diabetic Retinopathy Toxicol Pathol, October 1, 2006; 34(6): 763 - 775. [Full Text] [PDF]
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 R. Gupta, S. Karpatkin, and R. S. Basch Hematopoiesis and stem cell renewal in long-term bone marrow cultures containing catalase Blood, March 1, 2006; 107(5): 1837 - 1846. [Abstract] [Full Text] [PDF]
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A. P. Hall, F. R. Westwood, and P. F. Wadsworth Review of the Effects of Anti-Angiogenic Compounds on the Epiphyseal Growth Plate Toxicol Pathol, February 1, 2006; 34(2): 131 - 147. [Abstract] [Full Text] [PDF]
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 I. Cascone, L. Napione, F. Maniero, G. Serini, and F. Bussolino Stable interaction between {alpha}5{beta}1 integrin and Tie2 tyrosine kinase receptor regulates endothelial cell response to Ang-1 J. Cell Biol., September 12, 2005; 170(6): 993 - 1004. [Abstract] [Full Text] [PDF]
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M. R. Machein, A. Knedla, R. Knoth, S. Wagner, E. Neuschl, and K. H. Plate Angiopoietin-1 Promotes Tumor Angiogenesis in a Rat Glioma Model Am. J. Pathol., November 1, 2004; 165(5): 1557 - 1570. [Abstract] [Full Text] [PDF]
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Y. Xu, Y.-j. Liu, and Q. Yu Angiopoietin-3 Inhibits Pulmonary Metastasis by Inhibiting Tumor Angiogenesis Cancer Res., September 1, 2004; 64(17): 6119 - 6126. [Abstract] [Full Text] [PDF]
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 J.-X. Chen, Y. Chen, L. DeBusk, W. Lin, and P. C. Lin Dual functional roles of Tie-2/angiopoietin in TNF-{alpha}-mediated angiogenesis Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H187 - H195. [Abstract] [Full Text] [PDF]
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F. Fan, O. Stoeltzing, W. Liu, M. F. McCarty, Y. D. Jung, N. Reinmuth, and L. M. Ellis Interleukin-1{beta} Regulates Angiopoietin-1 Expression in Human Endothelial Cells Cancer Res., May 1, 2004; 64(9): 3186 - 3190. [Abstract] [Full Text] [PDF]
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 C. Daly, V. Wong, E. Burova, Y. Wei, S. Zabski, J. Griffiths, K.-M. Lai, H. C. Lin, E. Ioffe, G. D. Yancopoulos, et al. Angiopoietin-1 modulates endothelial cell function and gene expression via the transcription factor FKHR (FOXO1) Genes & Dev., May 1, 2004; 18(9): 1060 - 1071. [Abstract] [Full Text] [PDF]
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 C.-H. Cho, R. A. Kammerer, H. J. Lee, K. Yasunaga, K.-T. Kim, H.-H. Choi, W. Kim, S. H. Kim, S. K. Park, G. M. Lee, et al. Designed angiopoietin-1 variant, COMP-Ang1, protects against radiation-induced endothelial cell apoptosis PNAS, April 13, 2004; 101(15): 5553 - 5558. [Abstract] [Full Text] [PDF]
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G. Zadeh, B. Qian, A. Okhowat, N. Sabha, C. D. Kontos, and A. Guha Targeting the Tie2/Tek Receptor in Astrocytomas Am. J. Pathol., February 1, 2004; 164(2): 467 - 476. [Abstract] [Full Text] [PDF]
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 K. G. Peters, C. D. Kontos, P. C. Lin, A. L. Wong, P. Rao, L. Huang, M. W. Dewhirst, and S. Sankar Functional Significance of Tie2 Signaling in the Adult Vasculature Recent Prog. Horm. Res., January 1, 2004; 59(1): 51 - 71. [Abstract] [Full Text]
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O. Stoeltzing, S. A. Ahmad, W. Liu, M. F. McCarty, J. S. Wey, A. A. Parikh, F. Fan, N. Reinmuth, M. Kawaguchi, C. D. Bucana, et al. Angiopoietin-1 Inhibits Vascular Permeability, Angiogenesis, and Growth of Hepatic Colon Cancer Tumors Cancer Res., June 15, 2003; 63(12): 3370 - 3377. [Abstract] [Full Text] [PDF]
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L. Zhang, N. Yang, J.-W. Park, D. Katsaros, S. Fracchioli, G. Cao, A. O'Brien-Jenkins, T. C. Randall, S. C. Rubin, and G. Coukos Tumor-derived Vascular Endothelial Growth Factor Up-Regulates Angiopoietin-2 in Host Endothelium and Destabilizes Host Vasculature, Supporting Angiogenesis in Ovarian Cancer Cancer Res., June 15, 2003; 63(12): 3403 - 3412. [Abstract] [Full Text] [PDF]
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S. Hirakawa, Y.-K. Hong, N. Harvey, V. Schacht, K. Matsuda, T. Libermann, and M. Detmar Identification of Vascular Lineage-Specific Genes by Transcriptional Profiling of Isolated Blood Vascular and Lymphatic Endothelial Cells Am. J. Pathol., February 1, 2003; 162(2): 575 - 586. [Abstract] [Full Text] [PDF]
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T. A. Baudino, C. McKay, H. Pendeville-Samain, J. A. Nilsson, K. H. Maclean, E. L. White, A. C. Davis, J. N. Ihle, and J. L. Cleveland c-Myc is essential for vasculogenesis and angiogenesis during development and tumor progression Genes & Dev., October 1, 2002; 16(19): 2530 - 2543. [Abstract] [Full Text] [PDF]
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 S.M. DALLABRIDA and M.A. RUPNICK Vascular Endothelium in Tissue Remodeling: Implications for Heart Failure Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 417 - 428. [Abstract] [PDF]
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