* Department of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037; and Life Sciences Division, Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, California 94720
Angiogenesis is characterized by distinct
phenotypic changes in vascular endothelial cells (EC).
Evidence is provided that the Hox D3 homeobox gene
mediates conversion of endothelium from the resting to
the angiogenic/invasive state. Stimulation of EC with
basic fibroblast growth factor (bFGF) resulted in increased expression of Hox D3, integrin v
3, and the
urokinase plasminogen activator (uPA). Hox D3 antisense blocked the ability of bFGF to induce uPA and
integrin
v
3 expression, yet had no effect on EC cell
proliferation or bFGF-mediated cyclin D1 expression. Expression of Hox D3, in the absence of bFGF, resulted in enhanced expression of integrin
v
3 and
uPA. In fact, sustained expression of Hox D3 in vivo on
the chick chorioallantoic membrane retained EC in this
invasive state and prevented vessel maturation leading to vascular malformations and endotheliomas. Therefore, Hox D3 regulates EC gene expression associated
with the invasive stage of angiogenesis.
ANGIOGENESIS requires coordinate changes in endothelial cell morphology and gene expression. In
the resting vasculature, quiescent capillary endothelial cells are in contact with a laminin-rich basement
membrane (BM)1 and are nonmigratory. In response to
angiogenic stimuli, including cytokines such as basic fibroblast growth factor (bFGF), tumor necrosis factor Proteolysis of the BM, along with enhanced vascular
permeability which accompanies angiogenesis, facilitates
an influx of serum proteins including vitronectin, fibronectin, and fibrinogen, creating a provisional extracellular
matrix on which EC migrate. Although not typically expressed by quiescent resting EC, angiogenic EC express
high levels of integrin One class of transcriptional activators that have been
linked to extensive tissue remodeling are the homeobox
(Hox) genes. In addition to their role in embryonic development, the Hox genes have been shown to play a significant role in differentiation and gene expression in adult
tissues (Lawrence and Largman, 1992 Given the dramatic changes in cell-extracellular matrix
interactions, EC morphology, and gene expression that occur during angiogenesis, we investigated expression of
Hox genes and their role in endothelial cell behavior.
Cells and Culture Conditions
Experiments were conducted using primary human umbilical vein endothelial cells (HUVEC; Clonetics, San Diego, CA) or an immortalized HUVEC cell line (line 1998; American Type Culture Collection, Rockville,
MD). HUVECs were routinely cultured in M199 plus 20% FCS and
ECGS (Upstate Biotechnologies, Lake Placid, NY). The immortalized 1998 cell line was maintained in M199 plus 5% FCS. For experiments in
which bFGF was added, HUVECS were maintained in M199 plus 5% FCS, while the 1998 cell line was maintained in M199 with 2% serum. Recombinant human bFGF was purchased from Upstate Biotechnologies, and also kindly supplied by Dr. Judith Abraham (Scios Nova Inc., Mountain View, CA). For experiments using basement membrane, 1 × 106 cells
were seeded onto thick layers of Matrigel (Becton Dickinson, Bedford,
MA). Chick embryo fibroblast isolation and maintenance of the viral
packaging cell line Q4dh was performed as previously described (Stoker
and Bissell, 1988 Transfection of immortalized HUVEC or Q4dh cell lines was performed using calcium phosphate and stable transfectants selected using
800 or 400 µg/ml G418, respectively (Sigma Chemical Co., St. Louis, MO).
At least two independently selected pools of stably transfected cells were
used for subsequent analysis.
Isolation of HOX Genes Expressed in EC
RT-PCR of HOX genes was performed using total RNA from primary
HUVECs cultured in the presence or absence of BM. After reverse transcription with oligo-dT primers (Gene Amp; Perkin Elmer, Norwalk, CT),
DNA was amplified using degenerate primers against the conserved homeodomain sequences as described by Friedman et al. (1994): forward
primer 5 For quantitative PCR analysis of Hox D3 mRNA, 1 µg of total RNA
was reverse transcribed using oligo-dT primers and amplified for 30 cycles
at 95, 60, and 72°C with Hox D3 primers spanning a coding region to eliminate amplification of possible contaminating genomic DNA (forward
primer [bp 1682-1705] 5 Construction of Expression Vectors
Human genomic Hox D3 DNA was a gift from Y. Taniguchi (Tokai
School of Medicine, Bohseidai, Isehara, Kanagawu, Japan). A full length
Hox D3 cDNA was constructed by inserting the 415-bp PCR product,
generated as described above, between the Pst I site (bp 1899) and the
EcoRI site (bp 3853) in the genomic Hox D3. Expression vectors were
constructed by inserting either the genomic or Hox D3 cDNA between
the KpnI and BamHI sites of pcDNA3 mammalian expression vector (Invitrogen). Antisense expression vectors were constructed by inserting the
genomic DNA between the BamHI and HindIII sites of the pcDNA3 in
the antisense orientation. For controls, cells were transfected with empty
pcDNA3 vectors.
Chicken retroviral expression vectors were constructed by inserting either the Hox D3 genomic DNA or cDNA between the ClaI and BamHI
sites of the replication-defective retroviral vector CK (a gift from B. Vennstrom, European Molecular Biology Laboratory, Heidelberg, Germany). These vectors were then stably transfected into the packing cell
line Q4dh to generate infectious virus (Stoker and Bissell, 1988 RNA Isolation and Northern Blot Analysis
Total RNA was extracted using the Quick-Prep Kit (Qiagen, Santa Clara,
CA). For cells cultured on BM, cells were first detached from Matrigel using Matripserse (Becton Dickinson) and RNA isolated as described
above. 10 or 20 µg total RNA was run through 1% agarose formaldehyde
gels and transferred to Hybond-N membranes (Amersham Intl., Arlington Heights, IL) according to standard procedures. Membranes were hybridized using [32P]dCTP-labeled cDNA probes and exposed to Kodak
X-Omat film at Western Blots
Cultured cells were lysed in buffer containing 1 M NaCl, 10 mM Tris, pH
7.5, 1% Triton X-100 and aprotinin, PMSF, and leupeptin. Total protein
concentration was determined using the BCA reagent kit (Pierce, Rockford, IL), and a total of 10 µg was run on 10 or 12% SDS-PAGE under reducing conditions. Gels were transferred to Immobilon nylon membranes
(Millipore, Bedford, MA) and blocked in TBS plus 5% milk proteins.
Blots were probed using either 1:200 dilution of monoclonal anti-human
cyclin D1 (sc-246; Santa Cruz Biotechnologies, Santa Cruz, CA) or 10 µg/
ml polyclonal rabbit antihuman Adhesion Assays
Cells were removed by incubation with versene (0.5 M EDTA in PBS, pH
7.4) and resuspended in FBM media (Clonetics Corp., San Diego, CA)
supplemented with 0.2 M MnCl2. A total of 5 × 105 cells was seeded into
each well of 24-well culture dishes (Costar Corp., Cambridge, MA) that
had been previously coated for 1 h at 37°C with either heat-denatured
BSA (1%) or 10 µg/ml human fibrinogen (Sigma Chemical Co.). In some
cases 25 µg/ml of a function blocking anti-human BrdU Incorporation
To determine the effect of Hox D3 on cell proliferation, the rate of DNA
synthesis was established by measuring BrdU incorporation in control and
EC transfected with either Hox D3 or Hox D3 antisense expression vectors. After incubation for 4 or 12 h with 10 µM BrdU, cells were fixed with
70% ethanol and stained with an anti-BrdU kit (Boehringer Mannheim,
Indianapolis, IN) followed by staining with 0.5 µg/ml DAPI (4,6 diamidino-2-phenylindole; Sigma Chemical Co.). The percentage of BrdU-positive nuclei was determined by counting at least six different fields in each
of the cell types tested.
Chick Chorioallantoic Membrane Assay
All assays were performed in 10-d-old pathogen-free embryos (SPAFAS).
Briefly, the chorioallantoic membranes (CAMs) were prepared as previously described by Brooks et al. (1994) Immunohistochemistry
Sections were briefly fixed in 100% acetone and equilibrated with PBS.
After blocking in PBS plus 5% BSA, sections were incubated with either 5 µg/ml monoclonal antihuman In Situ Hybridization
Hox D3 sense and antisense digoxigenin-labeled RNA probes were generated by linearizing the vector pCR II (Invitrogen) containing the 415-bp
Hox D3 cDNA (generated by RT-PCR described above) and incubating
with T7 or SP6 RNA polymerase and digoxigenin-conjugated dUTP (Genius; Boehringer Mannheim). In situ hybridization was performed on 5-µm
cryosections mounted on siliconized slides (Sigma Chemical Co.). Sections were fixed in 4% paraformaldehyde for 20 min and dehydrated in
30, 70, and 100% ethanol for 2 min each and hybridized overnight at 45°C
with 800 ng/ml of Hox D3 riboprobes in 50% formamide, 10% dextran
sulfate, 1% blocking reagent (DIG nucleic acid detection kit; Boehringer
Mannheim), 300 µg/ml yeast tRNA (Sigma Chemical Co.), 3 mM NaCl, 10 mM Tris, pH 7.5, and 1 mM EDTA. After washing in 0.2× SSPE at 50°C
for 2 h, sections were incubated overnight with a 1:500 dilution of HRP-conjugated antibodies against digoxigenin and color developed using
NBT/BCIP (DIG nucleic acid detection Kit; Boehringer Mannheim).
Nonspecific hybridization was assessed using Hox D3 sense riboprobes.
Hox Gene Expression and the Angiogenic
Phenotype of EC
EC cultured on basement membrane adopt a distinct morphology reminiscent of capillaries in vivo (Fig. 1 A, + BM), withdraw from the cell cycle, and cease DNA synthesis within 24 h (Kubota et al., 1988
Hox D3 Induces To determine whether the expression of Hox D3 influences expression of genes associated with the angiogenic
phenotype of EC, a HUVEC cell line was stably transfected with a Hox D3 expression vector. Northern blot
analysis shows that HUVECs overexpressing Hox D3
have significantly higher levels of steady-state
The Hox D3-mediated changes in expression of integrin
The fact that angiogenesis is often accompanied by EC
proliferation prompted us to examine whether Hox D3
also influenced the rate of EC growth. We therefore compared DNA synthesis, as determined by BrdU incorporation, in control EC and EC transfected with Hox D3 or
Hox D3 antisense expression vectors. In contrast to the
Hox D3-induced changes in expression of integrin Hox D3 Mediates bFGF-induced Expression of The angiogenic cytokine bFGF leads to expression of both
uPA and
Although bFGF can also act as an endothelial cell mitogen, expressing Hox D3 antisense had no effect on bFGF's
ability to induce cyclin D1 in EC (Fig. 4 C). Thus Hox D3
appears to mediate bFGF-induced expression of Sustained Expression of Hox D3
In Vivo Results in Vascular Malformations and
Development of Endotheliomas
During the late stages of angiogenesis, integrin
These vascular malformations or endotheliomas ultimately resulted in the formation of large hemorrhagic zones
within the tumors generated by 15/18 Hox D3-infected embryos, but was not observed in tumors of the control embryos (Fig. 6, A and B). H&E staining of sectioned Hox
D3-infected tissue showed large endothelial-lined cysts filled
with hematopoietic cells, many of which had hemorrhaged (Fig. 6 D). In contrast, tumors producing control virus
showed normal tumor-induced angiogenesis (Fig. 6 C). These
findings not only establish a role for Hox D3 in EC function but also emphasize the requirement for tightly regulated
expression of Hox D3 during the early events of angiogenesis.
During embryonic development, normal tissue patterning
depends upon temporally and spatially restricted expression of Hox genes. Evidence is presented in this report
that patterning of the vasculature during angiogenesis is
affected by expression of the Hox D3 homeobox gene in
EC. We show that quiescent EC in contact with a BM express minimal levels of Hox D3, integrin Our findings that prolonged expression of Hox D3 results in abnormal vascular morphology also emphasizes
the requirement for tightly regulated expression of Hox
genes in EC. Previous studies have linked sustained expression of angiogenic mediators, including bFGF and
uPA with vascular tumors (Takahashi et al., 1994 Previous studies have linked FGF to the induction of
Hox D genes during embryonic limb development, although the precise targets of Hox D gene activity have not
been identified (Duprez et al., 1996 We have also shown that Hox D3 antisense blocks the
bFGF-induced expression of integrin Although both the integrin , and
vascular endothelial growth factor, vascular endothelial
cells (EC) reenter the cell cycle and upregulate proteolytic activity to degrade the existing BM, facilitating their invasion into stromal tissue. These vascular sprouts then synthesize a new BM and undergo morphological reorganization into mature quiescent, lumen-containing capillaries.
v
3, which can effectively bind this
provisional matrix (Brooks et al., 1994
; for review see
Cheresh and Mecham, 1994
). In fact, both matrix-degrading serine and metalloproteinases and integrin
v
3 have
been shown to play essential roles in new vessel formation,
as inhibition of their respective activities will impair tumor- or cytokine-mediated angiogenesis (Mignatti et al.,
1989
; Brooks et al., 1994
; Johnson et al., 1994
; Min et al.,
1996
). The mechanism by which angiogenic cytokines coordinately upregulate expression of proteases and adhesion molecules involved in angiogenesis however is not
known.
; Takeshita et al.,
1993
; Savageau et al., 1995
). Inappropriate Hox gene expression has also been linked to tumorigenic tissues (Friedmann et al., 1994
; Redline et al., 1994
). Hox genes
are characterized by a highly conserved 180-bp DNA-binding domain known as the homeodomain, which interacts directly with DNA to activate transcription of genes
(Desplan et al., 1985
). Putative target genes for Hox
DNA-binding proteins include other Hox genes and transcription factors, cell adhesion molecules, extracellular proteins, and growth factors (for reviews see Botas, 1993
;
and Edelman and Jones, 1993
; Immergluck et al., 1990
;
Goomer et al., 1994
; Taniguchi et al., 1995
). Interestingly,
in the genome, Hox and integrin receptor gene families
colocalize in clusters, indicative of parallel, coordinate
evolution, further supporting a link between these groups
of genes associated with tissue patterning (Wang et al.,
1995
).
MATERIALS AND METHODS
).
-GGAATTCCGARCTNGARAARGARTT-3
; and reverse
primer 5
-CCCAAGCTTRTTYTGRAACCADATYTT-3
. PCR reaction conditions include initial denaturation for 2 min at 95°C followed by
35 cycles at 95, 55, and 72°C for 1 min each plus a final incubation for 10 min at 72°C. In some instances, samples were reamplified for an additional
25 cycles using similar reaction conditions. Final PCR products were
cloned into TA cloning vectors (Invitrogen, San Diego, CA) and positive
clones amplified and Hox sequences identified by the dideoxy sequencing
method of Sanger et al. (1977)
.
-TGGTCTGAACTCAGAGCAGCAGC-3
and
reverse primer [bp 3962-3938] 5
-TCATGCGCCGGTTCTGGAACCA-3
). This yielded the predicted 415-bp PCR product, which was confirmed by DNA sequencing. To normalize for amounts of starting RNA, equal volumes of reaction mixtures were amplified for 35 cycles at 95, 60, and
72°C for 1 min each using primers for human GAPDH; forward primer
(bp 212-236) 5
-CCACCCATGGCAAATTCCATGGCA-3
; and reverse primer (bp 787-811) 5
-TCTAGACGGCAGGTCAGGTCCACC-3
;
(Stratagene, La Jolla, CA). PCR products were visualized and isolated by
running through 2% agarose gels containing 1 µg/ml ethidium bromide.
). Production of infectious retrovirus was monitored by infecting chick embryo fibroblasts with supernatants from cultures of stably transfected packing
cells as described by Stoker and Bissell (1988)
.
70°C. cDNA probes for human
3 and
5 integrin were
from E. Filardo (Brown University, Providence, RI). cDNA probe for human urokinase plasminogen activator (uPA) was kindly provided by Graham Parry (The Scripps Research Institute, La Jolla, CA).
3 integrin (AP-3) followed by a 1:1,000
dilution of either goat anti-rabbit or goat anti-mouse HRP (Biosource International, Camarillo, CA) and visualized by chemiluminescence (ECL;
Amersham Intl.).
v
3 integrin (mAb
LM609) were added along with cells. After extensive washing, cells were
fixed in 2% ethanol and stained with 1% crystal violet. The number of adherent cells was quantitated by absorbance at 600 nm.
. Q4dh virus-producing cells were
trypsinized, and a total of 5 × 106 cells were resuspended in 25 µl serum-free M199 and seeded onto the membranes and embryos incubated at
39°C. After 3 d, CAMs were harvested by washing in PBS and fixed for 30 min in 2% paraformaldehyde followed by washing in 0.1 M glycine/PBS.
CAMs were embedded into embedding medium (OCT; Miles, Elkhardt,
IN), flash frozen in liquid nitrogen, and stored at
80°C. 5-µm sections
were prepared and mounted on slides for immunohistochemistry.
v
3 (LM609) or a 1:1,000 dilution of polyclonal rabbit anti-human von Willebrand Factor (BioGenex, San Ramon,
CA). Goat anti-mouse-rhodamine or goat anti-rabbit-FITC (Biosource
International, Camarillo, CA) were used as secondary antibodies. Sections were subsequently mounted using Fluoromount (Southern Biotechnologies, Birmingham, AL).
RESULTS
). This "differentiated phenotype" contrasts the characteristic cobblestone
morphology associated with proliferating EC cultured in
the absence of BM (Fig. 1 A,
BM). RT-PCR of mRNA
from cultured EC detected the presence of a number of
Hox gene mRNA's including Hox A4, B3, B4, B5, and D3
(data not shown). Previous studies have linked Hox D3
with expression of
3 integrin mRNA in erythroleukemia cells (Tanaguchi et al., 1995). This
3 subunit is common
to integrin
v
3, which is highly expressed by angiogenic
EC (Brooks et al., 1994
), and prompted us to examine
whether Hox D3 expression was related to the expression
of
v
3 on EC. We therefore performed quantitative
RT-PCR analysis of EC cultured in the presence (+) or
absence (
) of BM for 36 h. As shown in Fig. 1 B, Hox D3
expression was detected in proliferating EC but not in quiescent EC cultured on BM. Northern and Western blot
analysis also shows that high levels of both
3 integrin
mRNA and protein were preferentially expressed by proliferating EC. Similarly, uPA mRNA was also highly expressed in proliferating but not quiescent EC (Fig. 1 C).
Thus, while quiescent EC do not express detectable levels
of Hox D3,
3 integrin, or uPA mRNA, EC in the absence
of BM express Hox D3 and genes associated with the angiogenic phenotype.
Fig. 1.
Expression of Hox D3, 3 integrin, and uPA in quiescent and proliferative EC. (A) Morphology of HUVEC cultured
in the presence (+) or absence (
) of reconstituted BM after 24 h
in M199 containing 5% FCS. (B) RT-PCR of 1 µg mRNA from
HUVEC cultured for 48 h with (+) or without (
) reconstituted
BM using primers for Hox D3 or GAPDH. (C) Western (top)
and Northern (bottom) blot analysis of integrin
3 expression in
HUVEC cultured in the presence (+) or absence (
) of BM for
48 h. Northern blots were reprobed for uPA mRNA expression.
The bottom panel shows ethidium bromide staining of total RNA
loaded in the corresponding gel.
[View Larger Versions of these Images (63 + 22K GIF file)]
3 Integrin and uPA mRNA but Does
Not Influence EC Proliferation
3 integrin and uPA mRNA levels as compared to cells transfected
with control vectors. In contrast, mRNA encoding integrin
5, which is not upreglated during angiogenesis, did not
differ in control or Hox D3-transfected cells (Fig. 2 A). To
establish whether Hox D3 was required for EC expression
of
3 integrin and uPA, HUVECs were stably transfected
with Hox D3 antisense expression vectors. Compared to
control-transfected cells, baseline levels of both
3 integrin and uPA mRNA were significantly reduced in cells
expressing Hox D3 antisense. In contrast, levels of
5 integrin mRNA were not influenced by expression of Hox D3
antisense (Fig. 2 B).
Fig. 2.
Influence of Hox D3 on 3 integrin and uPA expression in endothelial cells. (A) Northern blot analysis of
3 integrin,
5 integrin, uPA, and Hox D3 mRNA levels from 20 µg total RNA from immortalized HUVEC stably transfected with
control (C) or Hox D3 (Hox) expression vectors. The lower panel
shows ethidium bromide staining of total RNA loaded for each
sample in the corresponding gel. (B) Northern blot analysis of
3
integrin,
5 integrin, and uPA mRNA expression levels from immortalized HUVECS transfected with control (C) or antisense
expression vectors for Hox D3 (AS). RNA (20 µg) from each
sample was loaded, and total RNA was visualized by ethidium
bromide staining of the corresponding gel. The lower box shows
RT-PCR of 1 µg total RNA from cells transfected with control
(C) or antisense expression vectors against Hox D3 (AS) using
primers for Hox D3 or GAPDH.
[View Larger Version of this Image (38K GIF file)]
3 mRNA also resulted in corresponding changes in surface expression of functional
v
3 integrin. We compared
the ability of EC overexpressing Hox D3 or antisense
against Hox D3 to adhere to fibrinogen, a ligand for
v
3
on endothelial cells (Cheresh et al., 1989
). EC expressing
antisense against Hox D3 displayed significantly reduced capacity to adhere to fibrinogen, compared to control cells
(Fig. 3 A), whereas overexpressing Hox D3 increased EC
adhesion to fibrinogen (Fig. 3 A). Treatment of cells with
LM609, a function-blocking antibody against
v
3 (Cheresh,
1987
), resulted in a complete inhibition of the fibrinogen-mediated attachment of these cells (data not shown).
Thus, Hox D3 regulates the functional expression of
v
3
on endothelial cells.
Fig. 3.
Effects of Hox D3 sense and antisense expression on
EC adhesion and proliferation. (A) Adhesion to microtitre wells
coated with 10 µg/ml fibrinogen by EC transfected with Hox D3
sense () or antisense (
) expression vectors. Adhesion after 30 min was assessed by absorbance at 600 nm and expressed as a
percentage of adhesion displayed by control-transfected EC (n = 3). *P < 0.05. (B) BrdU incorporation in control-transfected EC
(
), Hox D3-overexpressing cells (
), or cells expressing antisense against Hox D3 (
). Cells were labeled for 4 or 12 h with
10 mM BrdU. The percentage of cells staining positive for BrdU
was assessed by counting at least six different fields containing a
total of at least 400 cells each.
[View Larger Versions of these Images (16 + 20K GIF file)]
3 and uPA described above, no differences in DNA synthesis
were observed between control EC, EC expressing Hox
D3, or antisense against Hox D3 (Fig. 3 B). Therefore,
while Hox D3 antisense can influence the expression of integrin
v
3 and uPA in EC it apparently does not directly
influence the proliferation of these cells.
3
Integrin and uPA but Not Cyclin D1 in EC
3 integrin in EC, which subsequently contribute
to the invasive properties of EC during the early stages of
angiogenesis (Moscatelli et al., 1985
; Enenstein et al.,
1992
; Brooks et al., 1994
; Sepp et al., 1994
). To determine
whether Hox D3 could influence the bFGF-mediated induction of integrin
v
3 and uPA, EC were made quiescent by culturing on BM and treating with or without
bFGF and analyzed for expression of mRNA encoding
these proteins. As shown in Fig. 4, exposure of HUVECs
to bFGF led to an increase in Hox D3 expression after 8 h,
which was accompanied by increased
3 integrin and uPA
steady-state mRNA levels by 24 h (Fig. 4, A and B). In
contrast, in EC expressing antisense against Hox D3, the
ability of bFGF to induce high levels of expression of
3
integrin and uPA mRNA expression was attenuated. Importantly, integrin
5 mRNA was not altered in either the
control or antisense-transfected cells in the presence or
absence of bFGF (Fig. 4, A and B).
Fig. 4.
Hox D3 mediates bFGF-induced expression of 3 integrin and uPA. (A) RT-PCR of 1 µg total RNA from HUVEC 24 h after
treatment with 20 ng/ml bFGF (+bFGF) using primers for Hox D3 or GAPDH. (B) Northern blot analysis of
3 integrin,
5 integrin,
and uPA mRNA levels in HUVEC transfected with control plasmid (C) or antisense against Hox D3 (AS) after 24 h with (+) or without (
) addition of 20 ng/ml bFGF. (C) Western blot of cyclin D1 in immortalized HUVEC transfected with control or antisense against
Hox D3. After 24 h in serum-free M199, samples were collected at 2, 4, and 8 h after addition of 20 ng/ml bFGF. 10 µg total cell lysate
from each time point was separated on 12% SDS-PAGE, transferred to nylon membranes, and probed with a polyclonal antibody
against human cyclin D1.
[View Larger Versions of these Images (30 + 40 + 47K GIF file)]
3 integrin and uPA but does not directly influence endothelial
cell cycle progression. This is consistent with the observation that Hox D3 antisense had no effect on EC proliferation (Fig. 3 B). These findings indicate that Hox D3 appears to influence EC genes important for invasion but not
proliferation.
v
3 and
uPA are down regulated as EC reestablish an intact BM
and form a lumen, suggesting that Hox D3 may be required only during the initial stages of angiogenesis. Maintaining expression of Hox D3 therefore might be expected
to prolong expression of an invasive phenotype and interfere with normal vascular maturation and/or remodelling.
To test this possibility, we constructed replication-defective avian retroviral vectors designed to constitutively express human Hox D3 in vivo. Transfection of a transformed viral packaging cell line Q4dh with Hox D3 proviral
vectors yielded a retrovirus capable of infecting proliferating embryonic chick cells and driving expression of human
Hox D3 in these cells. When grafted onto 10-d chick embryo CAM's, transformed virus-producing Q4dh cells generate solid tumors in these animals. The continued production of infectious virus leads to infection of adjacent,
proliferating cells including tumor-associated EC. As shown
in Fig. 5 A, maintenance of Hox D3 expression in these tissues caused blood vessel malformations, reminiscent of
endotheliomas, which appeared as abnormal capillary-like structures several times greater in diameter than large capillaries and were filled with hematopoietic cells. A majority of the EC and certain hematopoietic cells within these
endotheliomas remained positive for
v
3, providing evidence that Hox D3 was active in these tissues (Fig. 5 B).
To confirm the presence of Hox D3 in these vascular structures, we performed in situ hybridization using probes that
detect retrovirally produced human Hox D3 but do not detect endogenous chick Hox genes. Retroviral-mediated Hox
D3 expression was observed in a wide variety of tissues including epithelium, connective tissue, hematopoietic cells,
and endothelial cells (Fig. 5 C). In contrast to the widespread viral infection and expression of Hox D3,
v
3 integrin was only detected in EC and some hematopoietic
cells, suggesting this expression was highly specific.
Fig. 5.
Colocalization of
Hox D3 and v
3 integrin in
endotheliomas in vivo. (A)
H&E-stained cross-section
of tissue infected by Hox D3-expressing retrovirus shows
an abnormally large capillary-like structure filled with
erythrocytes. (B) Immunofluorescent staining with
LM609 against
v
3 in the
corresponding serial section
shows strong positive staining in both the endothelial
component (ec) of this vascular structure and in hematopoietic cells (h) contained within. (C) In situ hybridization of retrovirally expressed
Hox D3 in corresponding serial section showing widespread expression in epithelium (ep), endothelial cells
(ec), connective tissue (c),
and hematopoietic cells (h).
(D) Control in situ hybridization in a serial section performed with a sense riboprobe for Hox D3. Bar, 20 µm.
[View Larger Version of this Image (55K GIF file)]
Fig. 6.
Effect of retroviral-mediated expression
of Hox D3 in chick embryos. (A and B) Morphology of tumors generated 3 d after grafting of QT6
cells producing control or Hox D3-expressing retrovirus onto 10 d chick CAMs. (C and D) H&E
staining in tissue cross-sections from tumors (t)
and vasculature produced by cells shedding control or Hox D3-expressing retrovirus. Note the
large hemorrhagic region containing hematopoietic cells (h) adjacent to tumor tissue in the Hox
D3-infected tissue. Bar, 50 µm.
[View Larger Version of this Image (111K GIF file)]
DISCUSSION
v
3, and uPA.
However, exposure of EC to the angiogenic cytokine
bFGF leads to increased levels of Hox D3, resulting in the
functional expression of the angiogenic-promoting genes
integrin
3 and uPA in these cells.
; Hatva et
al., 1996
; Kraling et al., 1996
). In vivo, transgenic mice expressing polyoma middle T oncogene develop endothelial
tumors, and in culture, these transformed EC form cystic
vascular structures reminiscent of endothelioma, a phenotype that can be reversed by inhibiting uPA activity (Bautch
et al., 1987
; Montesano et al., 1990
). In addition, embryonic chick cells infected with v-src, a downstream effector
of FGF, show elevated uPA activity, and v-src infection of
chick endothelial cells in vivo results in endotheliomas
(Sullivan and Quigley, 1986
; Stoker et al., 1990
). Thus,
while increased uPA activity is involved in normal capillary remodeling, chronic expression driven by Hox D3
would be expected to prevent EC from establishing basement membranes, forming patent vessels with lumens and
resuming a quiescent, differentiated phenotype. Furthermore, the sustained expression of integrin av
3 provides the necessary means for EC to adhere, migrate, and survive in this angiogenic environment and thereby may enhance the activity of matrix-degrading serine and metalloproteinases, which interact with this integrin (Brooks et
al., 1996
; Strömblad et al., 1996
).
). In this report it is apparent that Hox D3 induction of integrin
v
3 is tissue
specific, since this integrin was not detected in epithelial
tissues expressing human Hox D3. In fact, endogenous
Hox D3 is expressed in human skin epithelium, yet this tissue does not express
v
3 (Detmer et al., 1993
; Brooks et
al., 1994
; Brown et al., 1994
; Gailit et al., 1994
). We also
observed that quiescent vessels in the skin do not express
Hox D3 (Boudreau, N., unpublished observations). However, EC and certain hematopoietic cells infected with
Hox D3-expressing retrovirus subsequently stained positively for
v
3. Hox D3 has previously been shown to induce expression of the
3 subunit of the
IIb
3 platelet adhesion receptor in cultured human erythroleukemia
cells which can be induced to differentiate to erythroid
cells (Taniguchi et al., 1995
). Thus, the ability of Hox D3
to mediate expression of the
3 integrin subunit may be
restricted to cells of the hematopoietic/angioblast lineage
which arise from common blood islands during embryogenesis (Risau, 1995
).
3 and uPA mRNA,
yet has no effect on bFGF-induced expression of cyclin D1
in these cells. Furthermore, we have shown that Hox D3
does not directly influence the rate of cell proliferation,
and together these results suggest that these aspects of angiogenesis are differentially regulated. These observations
are also supported by studies showing Hox D3 overexpression had no effect on cell proliferation (Taniguchi et al.,
1995
). Therefore in EC, Hox D3 appears to selectively
regulate the expression of genes that contribute to extracellular matrix remodeling during angiogenesis.
3 and uPA promoters contain several potential Hox-binding consensus sequences,
the complex relationship between Hox genes makes it difficult to predict whether Hox D3 acts alone or in combination with sequentially activated Hox genes to induce expression of these angiogenic effectors. For example, studies with compound mutants of Hox D3 and A3, suggests that paralogous Hox genes act in a synergistic manner to influence tissue patterning (Condie and Capecchi,
1994
). Similarly, activation or inhibition of the paralogous
Hox B3 gene, which is also expressed in EC, modifies expression of several other Hox genes including D3 (Faiella
et al., 1994
). Nonetheless, in EC, expression of Hox D3 is
sufficient to convert quiescent EC to an angiogenic/invasive state. This is supported by the observation that Hox
D3 antisense prevents bFGF-mediated expression of both
3 integrin and uPA in these cells. Our findings also reveal
that temporally restricted expression of Hox D3 is required for normal angiogenesis since prolonged expression of Hox D3 maintains the angiogenic/invasive EC phenotype, thereby disrupting normal vascular remodeling.
Received for publication 23 January 1997 and in revised form 16 July 1997.
Address all correspondence to Nancy Boudreau, Department of Anatomy, Medical College of Virginia, 1101 E. Marshall Street, Richmond, VA 23298. Tel.: (804) 828-7887. Fax: (804) 828-9477. E-mail: nboudrea @nsc.vcu.eduThe authors wish to thank Drs. Marlene Rabinovitch, Connie Myers, and especially Mina Bissell for critical reading of the manuscript and helpful discussions. We also thank Brian Eliceiri for help with graphics and Dr. Judith Abraham for providing bFGF used in these studies.
This work was supported by National Institutes of Health grant numbers HL54444 to D.A. Cheresh and Department of Energy grant number DEAC03-76-SF00098 to Mina J. Bissell, in whose laboratory this work was initiated. N. Boudreau was supported by a fellowship from the Joseph Drown Foundation. This is manuscript number 10482-IMM from the Scripps Research Institute.
bFGF, basic fibroblast growth factor; BM, basement membrane; CAM, chorioallantoic membrane; EC, vascular endothelial cells; HUVEC, human umbilical vein endothelial cells; uPA, urokinase plasminogen activator.
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