1 Center for Cardiovascular Research, University of Rochester School of Medicine
and Dentistry, Rochester NY 14642-8679, USA
2 Department of Neurobiology, Tottori University Faculty of Medicine, Yonago
683, Japan
3 Department of Pathology, Tottori University Faculty of Medicine, Yonago 683,
Japan
4 National Cardiovascular Center Research Institute, Osaka 565, Japan
Author for correspondence (e-mail: Keigi_Fugiwara{at}urmc.rochester.edu )
Accepted 21 December 2001
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Summary |
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Key words: angiogenesis, dystroglycan, endothelium, laminin
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Introduction |
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In general, however, cells use a number of different adhesion molecules to
attach to the ECM (Gumbiner,
1996), and it has long been known that VEC express non-integrin
types of laminin receptors whose identity still remains elusive
(Tressler et al., 1989
;
Basson et al., 1990
). On the
basis of its lamininbinding capacity, we have proposed dystroglycan (DG) to be
a non-integrin type of laminin receptor in VEC
(Shimizu et al., 1999
). DG is
the central component of the dystrophin-associated glycoprotein complex and
consists of
and ß subunits yielded by proteolytic cleavage of a
single precursor protein.
DG is a highly glycosylated extracellular
protein with a molecular mass of 120-190 kDa, and ßDG is a 43 kDa
transmembrane protein
(Ibraghimov-Beskrovnaya et al.,
1992
; Ibraghimov-Beskrovnaya
et al., 1993
). On the cell surface,
DG is non-covalently
anchored to ßDG (Yoshida et al.,
1994
). Originally identified in the skeletal muscle, DG is now
known to be expressed by a number of non-muscle cells. Elucidating its
functions in those cells is a subject of intense investigation
(Henry and Campbell, 1996
;
Henry and Campbell, 1998
).
By using antibody staining techniques, some investigators have reported DG
expression by VEC in blood vessels in human brain
(Uchino et al., 1996;
Yamamoto et al., 1997
) and by
cultured human umbilical endothelial cells
(Belkin and Smalheiser, 1996
).
However, DG expression in VEC is still a controversial issue. Tian et al.
(Tian et al., 1996
) reported
the absence of anti-DG staining in brain capillary endothelial cells, and
Durbeej et al. (Durbeej et al.,
1998
) suggested that the positive signals of anti-DG staining in
some blood vessels emanated from smooth muscle cells that expressed DG at a
high level. In our previous study (Shimizu
et al., 1999
), we demonstrated DG expression by cultured bovine
aortic endothelial cells (BAE) and, more importantly, we provided two lines of
evidence suggesting that DG was a cell adhesion molecule for VEC. First, BAE
adhesion to the laminin-1-coated surface was inhibited by soluble
DG
contained in the conditioned medium of cells transfected with DG cDNA. Second,
BAE adhesion was inhibited by a set of glycosaminoglycans (heparin, dextran
sulfate and fucoidan) that were shown to disrupt DGlaminin-dependent
cell adhesion in several other cell types
(Matsumura et al., 1997
).
In this study, we used BAE overexpressing full-length DG (FL-DG) or the
cytoplasmic domain of ßDG (ßDG) and provide direct evidence
for DG's role in VEC adhesion. These cells exhibited the phenotypes consistent
with our hypothesis that DG is a laminin-mediated cell adhesion molecule
(Shimizu et al., 1999
).
Furthermore, these cells showed negative correlation between the level of DG
expression and in vitro tube formation by VEC, suggesting a possible
regulatory function of DG in angiogenesis. When we studied DG expression in
VEC in vivo by immunohistochemistry, we found very low, if any, levels of
anti-DG staining in VEC of blood vessels in normal tissues. However, a high
level of staining was observed in VEC within the blood vessels found in
various tumors. Increased DG levels were also observed in the endothelium
within hyperplastic tissues. Our results suggest that DG has a role in
angiogenesis and indicate that DG expression is a good marker for the
angiogenic endothelium.
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Materials and Methods |
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Cell culture
The primary culture of BAE was obtained as described previously
(Sawamura et al., 1990). Cells
were routinely maintained in DMEM/10% FCS at 37°C in a humidified
atmosphere containing 5% CO2. They were passaged once a week at the
ratio of 1:5 to keep an exponentially growing state. All the cells were used
between the 5th and 10th passages.
Expression of full-length DG (FL-DG) and C-terminally truncated
ßDG (ßDG)
The entire coding sequence of the rabbit DG cDNA was subcloned into an
Epstein-Barr-Virus-based extra-chromosomal mammalian expression vector pREP9
(Invitrogen Lifetechnologies) to give pREP/F. A cDNA fragment encoding the
C-terminal cytoplasmic sequence (amino acid 775-895)
(Ibraghimov-Beskrovnaya et al.,
1992) was amplified by a polymerase chain reaction (PCR) and
subcloned into the same vector to give pREP/C. The pREP9 vector carries a gene
for geneticin resistance. The forward primer for PCR was designed to attach a
methionine residue on the N-terminus of the cytoplasmic domain. The PCR
product was verified by DNA sequencing. The protein product of pREP/C was
predicted to lack the transmembrane domain and to be freely soluble in the
cytoplasm.
For transient expression and immunostaining, BAE cultured on
poly-L-lysine-coated glass-bottom dishes were transfected with each plasmid
using lipofectoamine (Invitrogen Lifetechnologies) according to the
manufacturer's instructions. After a recovery period of 16 hours in DMEM/10%
FCS, the cells were double stained with Texas-Redphalloidin and
anti-ßDG as described previously
(Shimizu et al., 1999) in
order to determine transfection efficiency. Bound antibodies were detected
with Cy2-conjugated anti-mouse IgG (Amersham Pharmacia). To obtain enough
FL-DG/
ßDG-expressing cells for functional assays, cells were
transfected, and, after a recovery period, they were further incubated for 7
days in DMEM/10% FCS containing 0.4 mg/ml geneticin. To ascertain that all the
cells expressed FL-DG or
ßDG, we used immunofluorescence double
staining as described above. Fluorescence images were observed and recorded
using a Bio-Rad MRC1024 confocal microscope.
Flow cytometry
BAE were washed with phosphate-buffered saline (PBS), harvested by
trypsinization, fixed in 100% methanol for 10 minutes and then permeabilized
with 0.2% TritonX-100 for 10 minutes. They were stained with anti-ßDG (1
µg/ml in PBS/0.2% BSA) for 1 hour After washing by centrifugation and
resuspension, the cells were further incubated with Cy2-labeled anti-mouse IgG
and then subjected to flow cytometry using a FACScan flow cytometer (Becton
Dickinson Co., Mansfield, MA). DNA staining with propidium iodide and cell
cycle analyses were carried out as described
(Okazawa et al., 1998).
Western blotting
BAE were washed with PBS, lysed in the Laemmli's SDS sampling buffer. After
centrifugation at 1,000 g for 15 minutes to remove cell debris, the
protein concentration was determined using a BCA microprotein assay kit
(Pierce, Rockford, IL). Cell lysates were subjected to SDS-PAGE and western
transfer as described in Shimizu et al.
(Shimizu et al., 1999). To
obtain membrane and cytosolic fractions, BAE were washed with PBS, harvested
by scraping, resuspended in ice-cold lysis buffer (10 mM Tris-HCl pH 7.8, 1 mM
EGTA, 1 mM EDTA) and lysed by sonication. After centrifugation at 1,000
g for 15 minutes to remove cell debris, membrane and cytosolic
fractions were separated by centrifugation at 100,000 g for 30
minutes. The pellet was suspended in the lysis buffer, and the protein
concentration was determined using a BCA microprotein assay kit (Pierce,
Rockford, IL). The membrane and cytosolic fractions were subjected to SDS-PAGE
and western blotting (Shimizu et al.,
1999
).
Adhesion assay
96-well plates were coated with laminin-1 or fibronectin, and adhesion
assays were carried out as described
(Shimizu et al., 1999).
Briefly, BAE were seeded on the plates at a density of 2x104
cells/well in 100 µl of DMEM/0.2% BSA. After 2 hours of incubation in a
CO2 incubator, unattached cells were washed away with PBS and
attached cells were lysed in 100 µl TBS/0.2% Tween 20. The number of
attached cells in each well was estimated from a standard curve (cell number
versus lactic dehydrogenase activity in the lysates determined by using a
lactic dehydrogenase cytotoxic assay kit (Wako, Tokyo, Japan)). Unlike cells
cultured for 7 days that showed variable sizes
(Fig. 4A), these cells cultured
for only 2 hours exhibited the same cell size.
|
Migration assay
Cell migration was assessed by a wound healing assay on 35 mm dishes coated
with laminin-1. One million cells in 2 ml of DMEM/10% FCS were seeded in a
dish. Twenty-four hours later when the cells reached confluence, a linear
wound was made by scratching the monolayer with a 1 mm wide plastic scraper.
After washing with PBS, the cells were incubated in DMEM/0.2% BSA for up to 24
hours in a CO2 incubator, fixed by 4% paraformaldehyde in PBS and
stained with Coomassie blue.
Proliferation assay
BAE were seeded in 24-well plates at a density of 1.5x104
cells/well in 0.5 ml of DMEM/10% FCS. The medium was replaced every 2 days. At
the indicated times, the cells were harvested by trypsinization and the number
of cells were counted with a Coulter cell counter (Beckman Coulter, Fullerton,
CA).
Tube formation
48-well plates were coated with Matrigel (Becton Dickinson Labware)
according to the manufacturer's instruction. BAE were seeded at a density of
5x104 cells/well in 400 µl of DMEM/10% FCS and incubated
in a CO2 incubator up to 16 hours. At the indicated times, the
culture bed was photographed under a phase contrast microscope.
Immunohistochemistry
Human tissue sections of kidney cancer, colon cancer, peptic gastric ulcer
and nasal polyp were obtained. Anti-ßDG staining was performed on
formaldehyde-fixed, paraffin-embedded 4 µm thick sections using a Histofine
SAB-PO kit (Nichirei Co Ltd., Tokyo, Japan). The concentration of
anti-ßDG used was 1 µg/ml.
Statistical analysis
Where necessary, statistical analyses were done by ANOVA (analysis of
variance).
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Results |
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Flow cytometry was used to show that the geneticin selection method yielded
a homogeneous population of BAE for each case of transfection. After
transfection and selection, BAE were stained with anti-ßDG in suspension
and analyzed by flow cytometry. The average fluorescence intensity of
pREP/F-transfected cells was more than 10 times higher than that of the
vector-transfected cells (Fig.
1B). On the other hand, cells transfected with pREP/C were
indistinguishable from those transfected with the vector alone. Note that each
cell preparation forms a single peak, indicating that it consists of a
homogeneous population of cells. To investigate where in the cell these
exogenous proteins are present, we performed western blots on the membrane and
cytosolic fractions prepared from each of the transfected BAE. A dense 43 kDa
band reacting with anti-ßDG is the intact ßDG and was detected in
the membrane fraction of cells expressing the pREP/F product
(Fig. 1C; FL-DG). A low level
of full-length ßDG was present in the cytosol. It was not clear whether
the protein was originally in the cytoplasm or whther it was solubilized from
the membrane. In contrast, ßDG, detected as a 17 kDa protein, was
present exclusively in the cytoplasm and not in the membrane fraction. The 43
kDa band seen in the cells transfected with the vector alone (vector) or
pREP/C (
ßDG) is endogenous ßDG and was found only in the
membrane fraction. The level of endogenous ßDG was not affected by
overexpressing
ßDG (Fig.
1C). These results indicate that FL-DG is expressed on the cell
surface, presumably as an integral membrane protein, whereas
ßDG
is not associated with the cell membrane and instead is present in the
cytoplasm. To further characterize cells expressing fulllength DG (FL-DG) or
truncated ßDG (
ßDG), cell extracts were analyzed by
immunoblotting (Fig. 1D). The
control cells (vector) expressed
and ß DG as well as PECAM-1 and
utrophin (UTR). Dystrophin was not detected in the cell extract (data not
shown). Overexpression of FL-DG or
ßDG did not alter the
expression of PECAM-1 and utrophin. Thus, in a short-term experiment, the
expression levels of DG and utrophin, a DG-binding protein, are not tightly
coupled.
Effects of overexpressing FL-DG and ßDG on BAE adhesion
to laminin-1
We have shown that BAE adhesion to laminin-1 but not to fibronectin is, at
least partly, mediated by DG (Shimizu et
al., 1999). Using the same assay method and BAE overexpressing the
DG constructs, we were able to get further insights into the role of DG in
cell adhesion. FL-DG overexpression greatly enhanced cell adhesion to
laminin-1-coated dishes (Fig.
2). This adhesion was sensitive to heparin, a result consistent
with our previous study. FL-DG overexpression did not affect cell adhesion to
fibronectin-coated dishes, and heparin had no effect on the
fibronectin-mediated cell adhesion.
|
Overexpressing the cytoplasmic domain of ßDG inhibited adhesion of
cells to the culture dish coated with laminin-1but not with fibronectin
(Fig. 2). These results
indicate some functional role for the cytoplasmic portion of ßDG in
DG-mediated cell adhesion to laminin-1, although this part of the molecule
does not directly interact with the ligand. ßDG expression and the
addition of heparin together exerted the strongest inhibitory effect on cell
adhesion to laminin-1. However, there still existed a certain level of cell
adhesion activity after the simultaneous inhibition by
ßDG and
heparin, suggesting the possible presence of other laminin-1-binding
protein(s) on the cell surface. These results indicate that DG is a cell
adhesion molecule whose ECM ligand is laminin, that this cell adhesion is
achieved via the glycosaminoglycan on laminin and that the cytoplasmic domain
of ßDG plays a role in establishing cell adhesion.
Effects of FL-DG and ßDG expression on BAE migration on
laminin-1
The migratory activity of the cDNA-transfected cells on laminin-1 was
examined by an in vitro wound healing assay. Quantitative measurement of the
migration distance showed that the expression of the full-length DG did not
cause a discernible change in the migration distance within 24 hours of
wounding. In contrast, the migration of cells expressing ßDG was
significantly inhibited. The inhibitory effect was detectable within 6 hours
(not shown) and was considerable after 24 hours
(Fig. 3).
|
Effects of FL-DG and ßDG on BAE proliferation and cell
size
Long term effects of overexpressing the DG constructs were studied.
Transfected cells plated on regular culture dishes were maintained for several
days until they reached a post-confluent state.
Fig. 4A shows the monolayer
morphology of transfected cells. Although the vector-transfected BAE formed a
monolayer indistinguishable from that formed by untransfected cells, those
expressing FL-DG or ßDG exhibited drastically different morphology
of the monolayer.
Many of the FL-DG expressing cells appeared extended, revealing, on
average, a much larger cell outline (Fig.
4B). By contrast, cells expressing ßDG were densely
packed, showing a significantly smaller cell surface area. None of these
cultures showed signs of overlapping cells, indicating that BAE's ability to
from a tight monolayer was not affected by overexpression of the DG
constructs.
By eye, BAE expressing ßDG appeared densely packed, so we
obtained population growth curves for different cell types
(Fig. 4C). FL-DG-expressing
cells were significantly slower growing than the control cells although the
ßDG-expressing BAE showed faster growth. It appears that the
influence of DG on cell proliferation is to slow down, not to inhibit, cell
growth, as FL-DG cells were still able to grow, only at a reduced rate. Note
that, although the cells transfected with the vector or the cytoplasmic DG
fragment show contact inhibition, BAE overexpressing FL-DG are still growing.
Abnormal nuclear events, such as multi-nuclear cells and formation of giant
nuclei, were not commonly observed, suggesting that the processes of cell
division are not affected in these transfected cells.
Effects of FL-DG and ßDG on tube formation on
Matrigel
A hallmark of VEC is their ability to form a tube-like structure in vitro.
This is often referred to as in vitro angiogenesis. We investigated whether or
not our transfected BAE can form tube-like structures. Although there are
several systems for studying in vitro angiogenesis, we used the Matrigel
system because it gave us the most consistent results over a short period of
time. We chose this system also because the major component of Matrigel is
laminin-1, enabling us to assess the role of DG in angiogenesis as a laminin
receptor. The same number of cells was plated on Matrigel beds, and the extent
of tube formation was followed (Fig.
5). Both untransfected (not shown) and vector-transfected
(Fig. 5A, vector) BAE formed
small aggregates within 4 hours, and an elaborate network of cord structures
was found in the 8 and 16 hour cultures. One of the usual assumptions made to
explain the process of in vitro morphogenesis on Matrigel is increased roles
of cell-cell adhesion with concomitant weakening of cell-substrate adhesion.
DG being a laminin-based cell adhesion molecule, we thought that the
expression level of DG might be regulated during the process of in vitro tube
formation. To investigate this, we isolated membrane fractions from
untransfected BAE cultured on Matrigel at various times up to 24 hours and
assayed for ßDG expression levels. As shown in
Fig. 5B, DG expression was
downregulated during in vitro angiogenesis. The data suggest that DG is
inhibitory for angiogenesis in vitro and that the in vitro tube formation may
be inhibited by overexpressing DG. Indeed, BAE overexpressing DG failed to
form an extensive network of cord-like cell aggregates
(Fig. 5A; FL-DG). There was
little change in the overall appearance of the culture during the 16 hour
incubation period. Cells transfected with pREP/C, on the other hand, exhibited
an increased ability to form the cord structure
(Fig. 5A; ßDG).
These cells were able to form an extensive network of cords within 4 hours of
incubation. This result suggests that the cytoplasmic portion of ßDG can
effectively block the anti-angiogenic effect of endogenous DG.
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DG expression in in situ endothelium and angiogenesis
Our in vitro data have shown some interesting correlation between DG
expression and tube formation on Matrigel by BAE and suggested possible roles
of this laminin-binding protein in angiogenesis. To investigate if there is
correlation between angiogenesis and DG expression in vivo, we undertook an
immunohistochemical survey of DG expression within the endothelium of various
blood vessels found in human tissue sections. Since DG expression in in situ
VEC has been controversial, our initial studies were focused on anti-ßDG
staining of normal blood vessels. Studying normal vessels was also important
because our in vitro angiogenesis data predicted that normal (i.e.
nonangiogenic) vessels would express DG. A large number of blood vessels
within various nonpathological tissues was investigated, but in no cases did
we find strong anti-ßDG staining associated with VEC of vessels in normal
tissues. Other cell types, such as smooth muscle cells and various cells in
the connective tissue, were stained with anti-ßDG. Low levels of DG
expression in VEC cannot be excluded, but our survey appears to indicate that
DG expression in the endothelium of normal blood vessels is strongly
downregulated.
This study and some earlier reports, including ours, have demonstrated DG
expression by cultured VEC (Belkin and
Smalkeiser, 1996; Shimizu et
al., 1999
), indicating that these cells when isolated and placed
in the cell culture condition are induced to express DG. Although VEC in situ
and in vitro are different in many ways, one of the more fundamental
differences is their proliferation state. It is thought their proliferation
state that in situ VEC are growth arrested whereas those in vitro are growth
stimulated. To investigate whether DG expression is downregulated in vitro
when cell growth is inhibited, BAE were serum-arrested for 48 hours and
assayed for DG expression by immunoblots. We found that growth-arrested VEC
expressed DG at a significantly lower level than exponentially growing cells.
When the serum-starved cells were stimulated to grow, they went into the cell
cycle in a synchronous manner (Fig.
6A). Using anti-ßDG immunoblotting, we studied levels of DG
expression in such synchronous cultures. As shown in
Fig. 6B, the ßDG
expression level dramatically increased as the cells went into the S-phase. As
they passed through the S-phase, ßDG expression decreased. In fact, DG
expression appeared to cycle as cells traverse through the cell cycle. An
exponentially growing culture gave a high signal in the same immunoblot assay
(Fig. 6B; asynchronized).
|
Although most of the VEC in normal vessels are expected to be nonproliferating, we thought that vessels `under construction' might contain VEC that are still in the proliferative state. Such vessels may be found in various tumors. We obtained paraffin sections of surgical human specimens and performed anti-ßDG staining on them. Figs. 7A-F show blood vessels found in tissue sections of human kidney tumors. Since both normal and cancer tissues were present within the same section, blood vessels in normal tissue as well as those in the tumor were obtained from the same slide. The control vessels shown in Fig. 7A and Fig. 7C and vessels found in the malignant tissue (Fig. 7B,D) were from the same section. Another pair of normal (E) and tumor (F) tissues were obtained from a different specimen. Anti-ßDG stained many cells types within the normal kidney tissue, including the cells of the kidney tubules (Fig. 7A,E), the cells in the glomerulus (Fig. 7A) and smooth muscle cells of the artery (Fig. 7E; asterisk). However, VEC did not show strong staining. A venule present in Fig. 7A (boxed) is enlarged in Fig. 7C. Note that the cytoplasm of VEC (arrow) is light pink (the counter staining) and not associated with the brown DAB reaction product. Fig. 7E shows another example of an unstained VEC in normal venule from a different kidney tissue sample (arrow). In contrast, VEC of vessels found in the cancer tissue are strongly stained (Fig. 7B,D,F). Fig. 7G and Fig. 7H show arterioles found in a section of colon cancer. Smooth muscle cells of these vessels stained strongly with anti-ßDG. The arteriole in Fig. 7G is from the normal part of the tissue and shows no VEC staining (arrow). On the other hand the arteriole in Fig. 7H is in the cancer tissue within the same slide and shows strong endothelial staining (arrow).
|
To investigate whether or not anti-ßDG staining of VEC is a feature restricted to blood vessels in malignant tumors, we examined blood vessels within benign hyperplastic tissues, such as gastric ulcers and a nasal polyp. While the endothelium of the blood vessels in the normal (i.e. nonhyperplastic) area was not stained with anti-ßDG, vessels within the hyperplastic lesion contained VEC stained positively with the same antibody (data not shown). These immunohistochemical results suggest close correlation between the expression of DG in VEC and angiogenesis.
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Discussion |
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Laminin is an ECM protein, and our present and earlier studies have identified DG as a cell-substrate adhesion molecule whose ligand is laminin. Our current study shows that although the expression of ßDG, thus the DG complex, is practically undetectable in the endothelium of normal human tissues, it is highly upregulated in EVC in various tumors. Further analyses revealed strong anti-ßDG staining in VEC within hyperplastic tissues, indicating that DG expression in VEC in the adult body is not limited to tumors. These data collectively indicate strong correlation between DG expression and the endothelium of newly formed and/or forming blood vessels. This anti-ßDG staining was not dependent on the vessel type. To date, this is the most specific marker for angiogenic blood vessels.
We have attempted to elucidate the role of DG in angiogenesis by carrying
out in vitro experiments. We examined effects of DG expression on certain
physiological and phenotypic characteristics of VEC relevant to angiogenesis,
including cell proliferation, cell migration and tube formation. The results
of these experiments, however, do not converge neatly into a simple,
straightforward pattern that enables us to determine the role of DG in
angiogenesis at present. Our in vitro experiments indicate that ßDG is
needed for cells to effectively migrate on the laminin matrix and that
ßDG expression is suppressed in VEC made quiescent by serum-deprivation
and in those differentiated on Matrigel. These data are in line with the
pattern of DG expression in the body and enable us to interpret the
observations we made in pathological tissue preparations. However, the results
of the ßDG overexpression experiments suggest that lack of
effective signaling from ßDG is growth promoting and enhances
angiogenesis. These effects are the exact opposite of what one expects from
the in vivo DG expression pattern, where virtually no DG is expressed in VEC.
It is possible that a small but sufficient amount of DG is expressed, and this
hypothetical signaling is ongoing in normal vessels. Since the SAGE study
revealed significantly altered gene expression patterns between normal and
angiogenic VEC, involvement of other proteins needs to be considered. For
example, in normal vessels, it is possible that the expression of inhibitory
molecules for angiogenesis is upregulated or that molecules required for
angiogenesis are not expressed. It is also contrary to our expectation that
FL-DG expression was inhibitory for in vitro angiogenesis, albeit that only
the overexpression, but not the normal level of expression, of DG was
inhibitory. These results appears to suggest that although DG is a necessary
component of VEC for angiogenesis, its role in cell growth and tube formation
by VEC is to decelerate, not to inhibit totally, these activities. Although
interesting, it is not obvious at this time in what way these effects of DG
play roles in angiogenesis as well as in the whole body physiology. It is
anticipated that certain cellular activities need to be slowed down in order
for complex biological processes, such as angiogenesis, which require
coordination of a multiple number of events, to proceed in a controlled
manner.
It is interesting to note that cell migration on laminin was not affected
by FL-DG overexpression. By immunoblot analyses, we confirmed membrane
localization of the overexpressed DG, indicating that the molecule is capable
of binding to laminin. Given the fact that ßDG expression greatly
reduced cell migration, one may expect increased DG expression to increase
cell migration. However, this effect was not observed. Apparently, cultured
VEC already express enough of this cell adhesion molecule to provide the
maximum motile activity.
The function of DG is not well understood, particularly in nonmuscle cells.
The cytoplasmic tail of ßDG contains a proline-rich domain that is
necessary for its binding to dystrophin in skeletal muscle cells
(Jung et al., 1995). This
binding is thought to confer mechanical strength to the muscle sarcolemma
(Campbell, 1995
;
Henry and Campbell, 1996
). In
nonmuscle cells such as VEC, the same domain may bind to utrophin, the
nonmuscle homologue of dystrophin, and provide the similar reinforcement to
the plasma membrane. Studies on DG are hampered by the fact that DG gene
knockout in mice causes embryonic lethality
(Williamson et al., 1997
). As
an alternative, we expressed the soluble cytoplasmic tail of ßDG in VEC
to disrupt DG functions. Indeed, the expression of this construct gave VEC
`dominant negative' effects on their behaviors in vitro that are pertinent to
angiogenesis. We suggest that this approach is useful for analyzing DG
functions in the cell.
As an adhesion molecule, DG affected cells in both predictable and unique
ways. A decreased rate of proliferation in cells expressing FL-DG was
expected, as increased cell adhesion was shown to be inhibitory for cell
growth. For example, suppressed cell proliferation was noted in CHO cells
overexpressing an integrin subunit
(Giancotti and Ruoslathi,
1990) and in cancer cells whose E-cadherin-catenin system was
activated (Watabe et al.,
1994
). An unexpected observation was a significantly enlarged cell
outline of VEC overexpressing FL-DG. This effect was not VEC specific as the
same result was obtained using CHO cells (data not shown). This type of cell
enlargement does not occur in cells overexpressing an integrin subunit,
although it does cause a significant change in cell adhesion properties
(Giancotti and Ruoslahti,
1990
). A certain degree of cell spreading occurs when cell
adhesion property is increased, but the extent of cell enlargement is in
general far less than we observed in the cells overexpressing DG. The final
effect of a cell adhesion molecule on cells is unique for each molecule,
presumably because it activates a different set of signaling molecules inside
the cell. DG may be more deterministic of cell shape than integrins are.
In summary, our study demonstrates specific expression of DG in VEC of blood vessels in tumor and hyperplastic tissues, but the cause and effect relationship and the biological significance of this DG expression in the presumed angiogenic vessels remain unknown. Our study also demonstrate that changes in the DG expression level affect the behavior and phenotype of VEC, but the molecular mechanisms for these effects appear to be complex and need to be further investigated. The most important point of the study is that DG expression can be used as a specific marker of blood vessels in tumors. It may also serve as a marker for angiogenic blood vessels. For example, biopsy samples could be stained with anti-DG for diagnostic purposes. Surgically removed tissues may be stained with anti-DG to ascertain that cancerous tissue (in which blood vessels are positively stained) is surrounded by healthy tissue (that contains anti-DG-negative vessels). It may also be possible to use the DG expression as a molecular entryway to specifically inhibit blood vessel formation in tumors or to deliver drugs to tumors via specific endothelial cells expressing DG.
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