1 Center for Cell Biology and Cancer Research, Albany Medical College, Albany,
NY 12208, USA
2 The Center for Neuropharmacology and Neuroscience, Albany Medical College,
Albany, NY 12208, USA
3 The Institute for Vascular Health and Disease, Albany Medical College, Albany,
NY 12208, USA
4 The Center for Cardiovascular Sciences, Albany Medical College, Albany, NY
12208, USA
* Author for correspondence (e-mail: laflams{at}mail.amc.edu
Accepted 23 May 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Endothelial cells, Integrins, 6ß4, Angiogenesis, Development, Explant cultures, Schwann cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Integrins are /ß heterodimeric transmembrane proteins, and the
specific combination of
and ß subunits determines ligand-binding
specificity (Hynes, 1992
).
Endothelial cells express several different integrins, whose roles in
regulating endothelial cell adhesion, migration, proliferation and apoptosis
have been examined (Bazzoni et al.,
1999
; Hynes et al.,
1999
). The
v integrins have received most attention,
because they are upregulated during angiogenesis
(Brooks et al., 1994
;
Clark et al., 1996
) and
blocking their function with antagonists can inhibit angiogenesis
(Clark et al., 1996
;
Eliceiri and Cheresh, 1999
).
However, the role of
vß3 and
vß5 is more complex than
earlier thought, since mice lacking
3 and/or ß5 integrins show
normal vascular development and enhanced pathological angiogenesis, suggesting
that the
vß3 and
vß5 integrins may, in fact,
negatively regulate the angiogenic process
(Reynolds et al., 2002
). The
5ß1 integrin may play an important role in regulating new vessel
growth, as demonstrated by
5-null mice that die during embryogenesis as
a result of defects in vascular development
(Yang et al., 1993
). Blocking
the function of
5 integrins can also inhibit angiogenesis
(Kim et al., 2000
). Roles for
other integrin heterodimers in new vessel growth have also been described
(Bazzoni et al., 1999
;
Hynes et al., 1999
;
Senger et al., 1997
;
Senger et al., 2002
).
Additionally, several studies have reported the expression of
6ß4
in the vasculature (Enenstein and Kramer,
1994
; Kennel et al.,
1992
; Koukoulis et al.,
1991
; Ryynanen et al.,
1991
); however, there have been conflicting reports as to whether
it is expressed by endothelial cells or smooth muscle cells
(Cremona et al., 1994
).
Although the role of the 6ß4 integrin in the vasculature is
unclear, its function in epithelia is well characterized. In keratinocytes,
6ß4 is required for maintaining firm epithelial adhesion to the
underlying dermis (Dowling et al.,
1996
; van der Neut et al.,
1996
) by connecting the laminin-containing basement membrane with
the intracellular keratin intermediate filaments
(Borradori and Sonnenberg,
1999
). In some carcinoma cells, however,
6ß4 promotes
migration by activating specific signaling pathways and interacting with the
actin cytoskeleton (Mercurio et al.,
2001
; Trusolino et al.,
2001
). The function of
6ß4 in the vasculature may be
analogous to its function in epithelial and carcinoma cells;
6ß4
may confer a promigratory phenotype in response to angiogenic stimuli, and
also contribute to stable adhesion in the mature vasculature.
In this study, we were interested in determining whether the
6ß4 integrin functions in the vasculature to promote angiogenesis.
In initial studies, we confirmed the expression of
6ß4 on dermal
microvascular endothelial cells in situ. We did not observe expression of
6ß4 on smooth muscle cells. Additional experiments examined
whether the expression of
6ß4 was regulated during the formation
of new vessels. Explant angiogenesis assays, using segments of human saphenous
vein, demonstrated that endothelial cells downregulate
6ß4 in the
explant and that outgrowing endothelial cells also do not express
6ß4. Additionally, we did not observe expression of
6ß4 during early vascular development of the murine whisker pad,
but
6ß4 was expressed by the same vasculature of the adult animal.
The developmental expression of
6ß4 correlated spatially and
temporally with the maturation of the whisker pad and its corresponding
vasculature. Taken together, our studies indicate that
6ß4 is not
expressed during new vessel growth in our assays. This result implies the
6ß4 integrin may be a negative component of the angiogenic
switch.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of primary cells from human neonatal foreskin
Human neonatal foreskin, obtained under Albany Medical College IRB approved
protocols, was cut into small pieces (1x1 mm3) and
placed in 0.25% Trypsin in PBS for 16-20 hours at 4°C. The supernatant was
filtered through cheesecloth and centrifuged at 435 g at
4°C for 5 minutes. The cells were resuspended in PBS and plated into
6-well plates at 5-10x104 cells/well. Each well contained
coverslips precoated with poly-l-lysine (10 µg/ml in PBS for 2 hours at
37°C, washed 3x with PBS). The 6-well plates were centrifuged at 560
g for 5 minutes at 4°C, and the attached cells were fixed
with 4% paraformaldehyde in PBS for 30 minutes. The cells were washed 3x
with PBS, then immunostained.
Explant cultures
Explant cultures were prepared with slight modifications from a previously
published method (Nicosia and Madri,
1987; Nicosia and Ottinetti,
1990
). A segment of human saphenous vein, obtained under Albany
Medical College IRB approved protocols, was cut into cross sections
approximately 1 mm thick. The sections were washed 10x with PBS, and
then placed onto a solidified gel of fibrinogen (Sigma) in 24-well plates. The
gel was prepared by adding 3 mg of fibrinogen to 1 ml of EBM (Clonetics, San
Diego, CA), and gelled by the addition of 1 unit/ml of thrombin (Sigma). The
vein was positioned in the center of the well on top of the fibrinogen gel,
and overlaid with additional fibrinogen to which thrombin had been added
immediately prior to use. The gel was allowed to solidify, and EBM containing
15% serum, 100 units/ml penicillin/streptomycin, 1 µg/ml hydrocortisone
(Sigma), 10 ng/ml EGF (Collaborative Biomedical Products, Bedford, MA) was
added on top. The culture medium was changed every other day with additions of
the fibrinolytic inhibitor
-amino-n-caproic acid (Sigma) for the first 4
days at 300 µg/ml, then subsequently at 50 µg/ml. Human neonatal
foreskin explants were placed directly onto coverslips in 6-well plates, and
medium changed as above. Saphenous vein explants were fixed (as above) for 4
hours at 4°C then washed 3x for 5 minutes with PBS, cryoprotected in
30% sucrose in PBS overnight at 4°C, dehydrated by passage through 75% and
100% OCT for 24 hours each, and then cut into 10-12 µm sections. Human
saphenous vein, tissue and explant, exhibited high intensity broad-spectrum
autofluorescence and therefore we used immunoperoxidase staining, yielding
colored reaction products. Human foreskin explants were fixed for 30 minutes,
then immunostained.
Preparation of mouse mystacial (whisker) pads
Swiss Webster mice were purchased from Taconic. Adult, timed pregnancy mice
and neonates were euthanized and the mystacial pads were removed and
post-fixed and cryoprotected as for the explants. All procedures were in
accordance with Albany Medical College IACUC approved protocols.
Immunostaining
Sectioned tissue was rehydrated in PBS, permeabilized with 0.5% Triton
X-100 in PBS, blocked with 3% BSA-glycine, and then preincubated with 10%
normal goat serum (Pierce, Rockford, IL) in PBS. Sections were incubated
overnight at 4°C with primary antibodies diluted in PBS containing 0.5%
BSA, 10% goat serum and 0.1% Tween 20. Sections were washed and incubated with
the appropriately labeled fluorescent secondary antibodies, and then washed
and mounted in Anti-Fade (Molecular Probes). Cells on coverslips were
permeabilized, blocked with BSA-glycine, then incubated in primary antibody
for 1 hour at room temperature, but otherwise treated as above. For
immunoperoxidase staining, sections were rehydrated and endogenous peroxidase
quenched by incubating in 3% H2O2 in H2O for
10 minutes, then rinsed 2x for 1 minute in distilled H2O.
Sections were permeabilized and incubated with primary antibody and washed as
above, and then incubated with the appropriate biotinylated secondary
antibodies for 30 minutes, followed by incubation with Vectastain ABC Elite
reagents, and developed with the peroxidase substrate DAB reagents (Vector
Laboratories, Burlingame, CA) as described by the manufacturer. Sections were
dehydrated in a graded series of ethanol washes, cleared in xylene and mounted
with Permount (Fisher, Pittsburgh, PA). Controls were as above. In some
instances, tissue sections were rehydrated in PBS, incubated for 1 minute in
Gills Hematoxylin No. 2 (Polysciences, Warrington, PA), followed by 3 minutes
in tap water, 3 minutes in Scotts solution (24 mM NaHCO3, 166 mM
MgSO4 in H2O), 3 minutes in tap water, 10 seconds in
Eosin-Y (Richard Allen Scientific, Kalamazoo, MI), 3 minutes in tap water,
dehydrated and mounted as above. Immunostained material was visualized with
either an Olympus BX60 microscope with an attached Spot camera and associated
software, or with an Olympus AX70 with a Sony DKCST4 Digital Photo Camera
using Northern Eclipse software. Immunofluorescently labeled sections were
also visualized on a Noran Oz confocal laser scanning microscope interfaced
with a Nikon Diaphot 200 inverted microscope equipped with a PlanApo
x60, 1.4 NA oil-immersion objective. Controls for all immunofluorescent
staining in the absence of primary antibody showed no significant fluorescence
over tissue or cellular autofluorescence.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To more precisely characterize the expression of ß4 integrins on
vascular cells, individual cells isolated from trypsin-disrupted human
neonatal foreskin tissue were adhered to polylysine-coated coverslips and used
for immunocytochemistry. The cell-type expression of 6ß4 was
examined by determining whether ß4 was co-expressed with epithelial-,
endothelial-, and/or smooth muscle cell-specific markers. These studies
indicated that ß4 integrin was expressed by endothelial cells as
identified either by PECAM-1 (Fig.
1E) or vWF expression (data not shown). Some endothelial cells did
not show ß4 expression (not shown), which is consistent with published
findings that
6ß4 is expressed by a subset of vessels
(Mechtersheimer et al., 1994
).
We did not observe the co-expression of ß4 with smooth muscle actin
(Fig. 1G), suggesting that
ß4 may not be expressed by smooth muscle cells as previously reported
(Cremona et al., 1994
). As
expected only a subset of cytokeratin-positive cells expressed ß4
(Fig. 1I), since
6ß4 is known to be expressed by basal keratinocytes and not by
cells of the suprabasal layers (De Luca et
al., 1990
). Interestingly, the expression of ß4 remained
polarized in both endothelial cells and keratinocytes dissociated from
foreskin tissue (Fig.
1E,I).
The 6ß4 integrin is negatively regulated by angiogenic
endothelial cells in explant culture
To begin to understand the function of 6ß4 in the vasculature,
we investigated whether the expression of
6ß4 was regulated during
new vessel growth. To determine this, we performed explant angiogenesis assays
using segments of human saphenous vein
(Kruger et al., 2000
;
Slomp et al., 1996
) which have
been successfully used in these types of assays. When we examined ß4
expression in cross sections of saphenous vein, we found it was expressed by
endothelial cells of the vasa vasorum, but not by endothelial cells lining the
main lumen of the saphenous vein (Fig.
2A-E). To determine whether the expression of ß4 integrins is
regulated by outgrowing endothelial cells, human saphenous vein explants were
cultured in fibrinogen gels as described by others
(Kruger et al., 2000
;
Nicosia and Ottinetti, 1990
;
Slomp et al., 1996
).
Endothelial cell outgrowth was observed from the explant after 7 days with
robust growth observed after 14 days. At day 14, most of the cells were
arranged in tube-like structures that resembled vessels. Day-14 explants were
immunostained to confirm endothelial outgrowth with vWF
(Fig. 3A,B) and PECAM-1 (data
not shown). Virtually all cells in the outgrowth stained positive with vWF and
PECAM-1, with almost none of the outgrowth positive for smooth muscle actin
(Fig. 3E,F). Interestingly,
ß4 expression was not observed on outgrowing endothelial cells, or on
endothelial cells remaining in the vasa vasorum of the explant at day 14
(Fig. 3C,D). This was also true
of
6 (data not shown).
|
|
To exclude the possibility that the outgrowing endothelial cells were
originating only from the lumenal endothelial cells of the explant as opposed
to the ß4-positive endothelial cells present in the vasa vasorum, the
endothelial lining of the lumen was stripped by collagenase digestion, leaving
the endothelial cells of the vasa vasorum intact as determined by PECAM-1
staining (Fig. 4A) and vWF
(data not shown). Collagenase digestion did not affect the expression of
ß4 in the vasa vasorum (Fig.
4B). Explant assays were performed using collagenase-treated and
untreated saphenous vein segments. A phase image of day-14 explants that had
not been treated with collagenase showed normal outgrowth in the vessel lumen
(Fig. 4C). In contrast, there
was significantly diminished outgrowth into the lumen of the
collagenase-treated explant (Fig.
4D), whereas the outgrowth of endothelial cells from the vasa
vasorum of the vessel was unaffected (Fig.
4E). Immunostaining of collagenase-treated explants showed
endothelial-positive staining for PECAM-1
(Fig. 4F,G) and vWF (data not
shown). Similar to the outgrowth from untreated tissue, ß4 expression was
also downregulated both in the endothelial outgrowth and the original explant
at day 14 (Fig. 4H,I). This was
also true of 6 expression (data not shown). These results indicate that
endothelial cells downregulate
6ß4 expression during new vessel
growth in culture.
|
To confirm that the loss of endothelial expression of ß4 integrins was
due to cell-type specific regulation and was not an artifact of our outgrowth
assay, explants of human neonatal foreskin tissue were similarly cultured,
since migrating and proliferating basal keratinocytes are known to maintain
ß4 expression (Gipson et al.,
1993; Larjava et al.,
1993
; Mercurio et al.,
2001
; Nguyen et al.,
2000
). The outgrowing cells maintained an epithelial appearance,
and their identity as keratinocytes was confirmed by immunohistochemical
staining with an epithelial cytokeratin marker
(Fig. 4J). Dual labeling showed
that the outgrowing keratinocytes maintained their expression of ß4
integrins, which appeared in a punctate pattern typical of hemidesmosomes.
This result indicates that outgrowing endothelial cells in explant culture
down-regulate ß4 expression in a cell-type specific manner.
Expression of 6ß4 is temporally and spatially regulated
during vascular development
Since the expression of some genes can be altered when cells are placed in
culture (Antequera et al.,
1990; St Croix et al.,
2000
) and this could potentially occur in a cell-type-dependent
manner, we were interested in determining whether
6ß4 was
expressed by newly forming vessels in situ. To accomplish this, we analyzed
the expression of
6ß4 in newly forming vasculature using the
developing murine mystacial (whisker) pad as a model. This model was chosen
because the whisker pad has a well described and predictable vascular
architecture shown schematically in Fig.
5A (Fundin et al.,
1997b
). Initial studies demonstrated the expression of ß4
integrins in the vasculature of the adult whisker pad (Figs
5,
8). Because ß4 may be
expressed on endothelial cells, perineural cells and Schwann cells within the
dermis (Niessen et al., 1994
)
and because of limitations in the availability of immunological reagents for
murine tissue, we analyzed the expression of
6ß4 by
double-immunofluorescence staining of neighboring sections, using either
antibodies to PECAM-1 and to s100, a marker for Schwann cells
(Kligman and Hilt, 1988
;
Zimmer et al., 1995
), or
antibodies to ß4 and s100. [Note that although s100 expression is
concentrated in Schwann cells, it also labels perineurium, fat cells, and
chondrocytes to varying degrees (Zimmer et
al., 1995
).] Endothelial staining with PECAM-1 antibodies was
observed throughout the different vascular beds of the whisker follicle
(Fig. 5B). This staining was
distinct, compared to s100 expression. In the neighboring section, ß4
staining was prominent on the basal keratinocytes and epithelial invaginations
of both hair and whisker follicles, which served as a positive control
(Fig. 5C). ß4 was also
expressed by the Schwann cells and perineurium of nerves that were identified
by s100 labeling. ß4 was expressed on endothelial cells of most vessels
supporting the various whisker pad sites
(Fig. 5C,E,I). This was
confirmed by double-label immunofluorescence showing the colocalization of
ß4 and PECAM-1 (Fig.
8A-C). Interestingly, ß4 was lacking on a set of vessels
affiliated with papillary muscle slings
(Fig. 5G).
|
|
To examine ß4 expression during the development of the vasculature, the whisker pad from E19.5 embryos (where E20 corresponded with parturition) was analyzed similarly to the adult tissue shown in Fig. 5. In general, ß4 expression was not detected in the embryonic microvasculature when neighboring sections were stained with antibodies to PECAM-1 and ß4. However, some endothelial ß4 expression was observed in the caudal regions (Fig. 6A,B) and deep vasculature (Fig. 6C,D). Double labeling with ß4 and vWF supported this observation (data not shown). ß4 was also not coexpressed with s100, indicating that ß4 is not expressed by Schwann cells at this time during the development of the whisker pad (Fig. 6A,B).
|
Induction of ß4 expression in the whisker pad vasculature was analyzed at postnatal day (P), zero (P0, equivalent to E20), P3 and P7. At P0, ß4 expression could be seen on the vasculature in the caudal most region, and also in deeper regions of the tissue (Fig. 7B). However, at this time point, large regions of the vasculature still remained negative for ß4 expression, such as between all of the whisker follicles (Fig. 7A,B). At P3, ß4 expression was observed in the capillaries that lie between the facial muscles, again caudal and deep to the developing whisker pad (Fig. 7C,D). Using double-label immunofluorescence for ß4 and PECAM-1 expression, little or no detectable endothelial ß4 expression was observed on vessels between the whisker follicles (not shown) or between developing hair follicles in the upper dermis (Fig. 8D,E,F). By P7, the vasculature associated with the follicles of the whisker pad had started to express ß4 (Fig. 7E,F). It should also be noted that the expression of ß4 in the peripheral nervous system (perineural sheaths and Schwann cells) and ß4 expression in the vasculature showed a similar pattern of progression from caudal to rostral and deep to superficial regions during the development of the whisker pad (Fig. 7B). Together these data suggest that the temporal and spatial progression of the ß4 integrin in the vasculature may correlate with vascular maturation in the whisker pad.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanisms regulating the expression of 6ß4 in endothelial
cells are poorly understood. Our data suggests that signals known to drive the
angiogenic process, such as hypoxia, basic fibroblast growth factor (bFGF) and
vascular endothelial growth factor (VEGF) signaling
(Carmeliet, 2000
), may
negatively impact on the expression of
6ß4 in endothelial cells.
In contrast, the expression of the
v,
1 and
2 integrins
is positively regulated by angiogenic signals
(Brooks et al., 1994
;
Senger et al., 1997
). This is
likely to be physiologically important because endothelial cell migration and
invasion associated with angiogenesis is believed to occur through a collagen
I-rich matrix (Senger et al.,
1997
) or a provisional matrix rich in fibronectin and vitronectin,
which are ligands for these integrins
(Senger, 1996
). Interestingly,
bovine adrenal cortex endothelial cells upregulate the expression of
6ß4 in culture when stimulated with bFGF, suggesting that
6ß4 may also promote angiogenesis in a tissue-specific manner
(Klein et al., 1993
). This is
consistent with the observation that matrices rich in laminin 1, a known
ligand for
6ß4, induce the morphological differentiation of
endothelial cells into capillary-like structures in culture
(Grant and Kleinman,
1997
).
Our data further suggests that signals promoting vessel maturation may
positively regulate the expression of 6ß4 by endothelial cells.
For example, angiogenic vessels are known to undergo stabilization and
maturation by mechanisms that require interaction between endothelial and
mural cells, and the localized activation of TGFß, resulting in the
production of extracellular matrix components by endothelial cells
(Folkman and D'Amore, 1996
;
Hellstrom et al., 2001
;
Hirschi and D'Amore, 1996
;
Neubauer et al., 1999
;
Nicosia and Madri, 1987
;
Shanker et al., 1999
). Future
studies in our laboratory will address these questions.
The expression of 6ß4 by mature vessels, and its absence in
newly developing vessels suggests that
6ß4 may only be required
after vessel maturation. This is supported by the fact that
6ß4
integrin is a receptor for several laminin isoforms
(Borradori and Sonnenberg,
1999
; Lee et al.,
1992
), and although the isoforms expressed in the vasculature are
not fully characterized, the endothelial basement membrane is known to be rich
in laminins (Colognato and Yurchenco,
2000
). Thus,
6ß4 may be required for endothelial
adhesion to the underlying basement membrane to promote the integrity of
mature vessels. Consistent with this notion, previous studies from our
laboratory suggest that
6ß4 may uniquely contribute to endothelial
cell adhesion by forming a transmembrane link between the basement membrane
and the vimentin intermediate filament cytoskeleton
(Homan et al., 1998
).
The endothelial-specific downregulation of 6ß4 during
endothelial outgrowth contrasts with the known role of
6ß4 in
certain carcinoma cells where it promotes migration and invasion
(Gambaletta et al., 2000
;
Mercurio et al., 2001
;
Trusolino et al., 2001
). Basal
keratinocytes also maintain expression of
6ß4 during
re-epithelialization (Gipson et al.,
1993
; Larjava et al.,
1993
; Mercurio et al.,
2001
; Nguyen et al.,
2000
). These differences in cell-type specific regulation of
6ß4 may reflect the essential requirement of the epidermal layer
to be firmly attached to the underlying basement membrane by the basal
keratinocytes through their expression of
6ß4. This is illustrated
by the extensive detachment of the epidermal-dermal interface in neonatal
ß4 knockout mice (Dowling et al.,
1996
; van der Neut et al.,
1996
), and the microblistering phenotype observed in the skin of
patients with Epidermolysis Bullosa, which is the result of mutations in
ß4 (Pulkkinen and Uitto,
1999
). Together this strongly implies that even temporary
downregulation of
6ß4 expression by basal keratinocytes during
re-epithelialization may compromise skin integrity, whereas remodeling vessels
do not have this same requirement for maintained
6ß4
expression.
Also, in contrast to keratinocytes, Schwann cells do not express ß4
during in vitro migration except when they begin ensheathing/myelinating axons
(Einheber et al., 1993;
Niessen et al., 1994
;
Previtali et al., 2001
). Our
data indicates that ß4 is not expressed by Schwann cells during embryonic
development of the whisker pad, but is detected on Schwann cells at P0, as
reported by others (Feltri et al.,
2002
). This expression pattern of ß4 on perineural cells and
Schwann cells, appears similar temporally and spatially to that observed on
endothelial cells. Thus, similar signaling and/or transcriptional mechanisms
may regulate ß4 expression in Schwann cells and endothelial cells.
Our findings that 6ß4 is not expressed on newly forming vessels
is consistent with, and may in fact help explain, the lack of a gross
pathological phenotype in the vasculature of the ß4-null mice
(Dowling et al., 1996
;
van der Neut et al., 1996
).
Our results also suggest that endothelial expression of
6ß4 may be
a negative component of angiogenesis, and that its expression needs to be
downregulated at the onset of new vessel growth. This is consistent with
recent reports that suggested that some integrins may negatively modulate
angiogenic processes (Reynolds et al.,
2002
; Stupack et al.,
2001
), perhaps by inhibiting endothelial growth and even
triggering apoptosis when the appropriate ligands are unavailable.
Additional studies are needed to fully understand the role of
6ß4 in the vasculature. A vascular-specific conditional knockout
would be valuable in this regard. Even with this in hand, if
6ß4
functions together with other adhesion molecules to promote vascular
integrity, a vascular phenotype may only be observed after vascular challenge,
such as intense cardiovascular activity or an inflammatory response. Based on
our findings, we propose a novel function for
6ß4 as a potential
negative regulator of angiogenesis in some instances. Future experiments will
test this hypothesis by determining whether forced vascular expression of
ß4 integrins significantly inhibits angiogenesis and/or vascular
development. Additionally, it will be important to determine whether
6ß4 is also negatively regulated during angiogenesis associated
with wound healing and tumor vascularization, since normal and pathological
angiogenesis can involve different mechanisms
(Carmeliet et al., 2001
;
Carmeliet and Jain, 2000
;
St Croix et al., 2000
).
Finally, if expression of ß4 can be anti-angiogenic, it will become
important to determine the molecular mechanisms involved, since such studies
may have potential therapeutic value in controlling pathological angiogenesis
in specific tissue environments.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albelda, S. M., Muller, W. A., Buck, C. A. and Newman, P. J. (1991). Molecular and cellular properties of PECAM-1 (endo CAM/CD31): a novel vascular cell-cell adhesion molecule. J. Cell Biol. 114, 1059-1068.[Abstract]
Antequera, F., Boyes, J. and Bird, A. (1990). High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell 62, 503-514.[Medline]
Bazzoni, G., Dejana, E. and Lampugnani, M. G. (1999). Endothelial adhesion molecules in the development of the vascular tree: the garden of forking paths. Curr. Opin. Cell Biol. 11, 573-581.[CrossRef][Medline]
Borradori, L. and Sonnenberg, A. (1999).
Structure and function of hemidesmosomes: more than simple adhesion complexes.
J. Invest. Dermatol.
112,
411-418.
Brooks, P. C., Clark, R. A. and Cheresh, D. A.
(1994). Requirement of vascular integrin vß3 for
angiogenesis. Science
264,
569-571.[Medline]
Carmeliet, P. (2000). Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6, 389-395.[CrossRef][Medline]
Carmeliet, P. and Jain, R. K. (2000). Angiogenesis in cancer and other diseases. Nature 407, 249-257.[CrossRef][Medline]
Carmeliet, P., Moons, L., Luttun, A., Vincenti, V., Compernolle, V., De Mol, M., Wu, Y., Bono, F., Devy, L., Beck, H. et al. (2001). Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat. Med. 7, 575-583.[CrossRef][Medline]
Clark, R. A., Tonnesen, M. G., Gailit, J. and Cheresh, D. A.
(1996). Transient functional expression of vß3 on
vascular cells during wound repair. Am. J. Pathol.
148,
1407-1421.[Abstract]
Colognato, H. and Yurchenco, P. D. (2000). Form and function: the laminin family of heterotrimers. Dev. Dyn. 218, 213-234.[CrossRef][Medline]
Cremona, O., Savoia, P., Marchisio, P. C., Gabbiani, G. and
Chaponnier, C. (1994). The 6ß4 integrin subunits
are expressed by smooth muscle cells of human small vessels: a new
localization in mesenchymal cells. J. Histochem.
Cytochem. 42,
1221-1228.
De Luca, M., Tamura, R. N., Kajiji, S., Bondanza, S., Rossino, P., Cancedda, R., Marchisio, P. C. and Quaranta, V. (1990). Polarized integrin mediates human keratinocyte adhesion to basal lamina. Proc. Natl. Acad. Sci. USA 87, 6888-6892.[Abstract]
Dowling, J., Yu, Q. C. and Fuchs, E. (1996). ß4 integrin is required for hemidesmosome formation, cell adhesion and cell survival. J. Cell Biol. 134, 559-572.[Abstract]
Einheber, S., Milner, T. A., Giancotti, F. and Salzer, J. L.
(1993). Axonal regulation of Schwann cell integrin expression
suggests a role for 6ß4 in myelination. J. Cell
Biol. 123,
1223-1236.[Abstract]
Eliceiri, B. P. and Cheresh, D. A. (1999). The
role of v integrins during angiogenesis: insights into potential
mechanisms of action and clinical development. J. Clin.
Invest. 103,
1227-1230.
Enenstein, J. and Kramer, R. H. (1994). Confocal microscopic analysis of integrin expression on the microvasculature and its sprouts in the neonatal foreskin. J. Invest. Dermatol. 103, 381-386.[Abstract]
Feltri, M. L., Porta, D. G., Previtali, S. C., Nodari, A.,
Migliavacca, B., Cassetti, A., Littlewood-Evans, A., Reichardt, L. F.,
Messing, A., Quattrini, A., Mueller, U. and Wrabetz, L.
(2002). Conditional disruption of ß1 integrin in Schwann
cells impedes interactions with axons. J. Cell Biol.
156,
199-210.
Flamme, I., Frolich, T. and Risau, W. (1997). Molecular mechanisms of vasculogenesis and embryonic angiogenesis. J. Cell. Physiol. 173, 206-210.[CrossRef][Medline]
Folkman, J. and D'Amore, P. A. (1996). Blood vessel formation: what is its molecular basis? Cell 87, 1153-1155.[Medline]
Fundin, B. T., Arvidsson, J., Aldskogius, H., Johansson, O., Rice, S. N. and Rice, F. L. (1997a). Comprehensive immunofluorescence and lectin binding analysis of intervibrissal fur innervation in the mystacial pad of the rat. J. Comp. Neurol. 385, 185-206.[CrossRef][Medline]
Fundin, B. T., Pfaller, K. and Rice, F. L. (1997b). Different distributions of the sensory and autonomic innervation among the microvasculature of the rat mystacial pad. J. Comp. Neurol. 389, 545-568.[CrossRef][Medline]
Gambaletta, D., Marchetti, A., Benedetti, L., Mercurio, A. M.,
Sacchi, A. and Falcioni, R. (2000). Cooperative signaling
between 6ß4 integrin and ErbB-2 receptor is required to promote
phosphatidylinositol 3-kinase-dependent invasion. J. Biol.
Chem. 275,
10604-10610.
Giancotti, F. G. and Ruoslahti, E. (1999).
Integrin signaling. Science
285,
1028-1032.
Gipson, I. K., Spurr-Michaud, S., Tisdale, A., Elwell, J. and
Stepp, M. A. (1993). Redistribution of the hemidesmosome
components 6ß4 integrin and bullous pemphigoid antigens during
epithelial wound healing. Exp. Cell. Res.
207, 86-98.[CrossRef][Medline]
Grant, D. S. and Kleinman, H. K. (1997). Regulation of capillary formation by laminin and other components of the extracellular matrix. EXS 79, 317-333.[Medline]
Hellstrom, M., Gerhardt, H., Kalen, M., Li, X., Eriksson, U.,
Wolburg, H. and Betsholtz, C. (2001). Lack of pericytes leads
to endothelial hyperplasia and abnormal vascular morphogenesis. J.
Cell Biol. 153,
543-553.
Hirschi, K. K. and D'Amore, P. A. (1996). Pericytes in the microvasculature. Cardiovasc. Res. 32, 687-698.[CrossRef][Medline]
Homan, S. M., Mercurio, A. M. and LaFlamme, S. E.
(1998). Endothelial cells assemble two distinct
6ß4-containing vimentin-associated structures: roles for ligand
binding and the ß4 cytoplasmic tail. J. Cell Sci.
111,
2717-2728.
Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25.[Medline]
Hynes, R. O., Bader, B. L. and Hodivala-Dilke, K. (1999). Integrins in vascular development. Braz. J. Med. Biol. Res. 32, 501-510.[Medline]
Kennel, S. J., Godfrey, V., Ch'ang, L. Y., Lankford, T. K., Foote, L. J. and Makkinje, A. (1992). The ß4 subunit of the integrin family is displayed on a restricted subset of endothelium in mice. J. Cell Sci. 101, 145-150.[Abstract]
Kennel, S., Foote, L. and Flynn, K. (1986). Tumor antigen on benign adenomas and on murine lung carcinomas quantitated by a two-site monoclonal antibody assay. Cancer Res. 46, 707-712.[Abstract]
Kim, S., Bell, K., Mousa, S. A. and Varner, J. A.
(2000). Regulation of angiogenesis in vivo by ligation of
integrin 5ß1 with the central cell-binding domain of fibronectin.
Am. J. Pathol. 156,
1345-1362.
Klein, S., Giancotti, F. G., Presta, M., Albelda, S. M., Buck, C. A. and Rifkin, D. B. (1993). Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells. Mol. Biol. Cell 4, 973-982.[Abstract]
Kligman, D. and Hilt, D. C. (1988). The S100 protein family. Trends Biochem. Sci. 13, 437-443.[CrossRef][Medline]
Koukoulis, G. K., Virtanen, I., Korhonen, M., Laitinen, L., Quaranta, V. and Gould, V. E. (1991). Immunohistochemical localization of integrins in the normal, hyperplastic, and neoplastic breast. Correlations with their functions as receptors and cell adhesion molecules. Am. J. Pathol. 139, 787-799.[Abstract]
Kruger, E. A., Duray, P. H., Tsokos, M. G., Venzon, D. J., Libutti, S. K., Dixon, S. C., Rudek, M. A., Pluda, J., Allegra, C. and Figg, W. D. (2000). Endostatin inhibits microvessel formation in the ex vivo rat aortic ring angiogenesis assay. Biochem. Biophys. Res. Commun. 268, 183-191.[CrossRef][Medline]
Larjava, H., Salo, T., Haapasalmi, K., Kramer, R. H. and Heino, J. (1993). Expression of integrins and basement membrane components by wound keratinocytes. J. Clin. Invest. 92, 1425-1435.[Medline]
Lee, E. C., Lotz, M. M., Steele, G. D., Jr and Mercurio, A.
M. (1992). The integrin 6ß4 is a laminin
receptor. J. Cell Biol.
117,
671-678.[Abstract]
Mechtersheimer, G., Barth, T., Hartschuh, W., Lehnert, T. and Moller, P. (1994). In situ expression of ß1, ß3 and ß4 integrin subunits in nonneoplastic endothelium and vascular tumours. Virchows Arch. 425, 375-384.[Medline]
Mercurio, A. M., Rabinovitz, I. and Shaw, L. M.
(2001). The 6ß4 integrin and epithelial cell
migration. Curr. Opin. Cell Biol.
13,
541-545.[CrossRef][Medline]
Neubauer, K., Kruger, M., Quondamatteo, F., Knittel, T., Saile, B. and Ramadori, G. (1999). Transforming growth factor-ß1 stimulates the synthesis of basement membrane proteins laminin, collagen type IV and entactin in rat liver sinusoidal endothelial cells. J. Hepatol. 31, 692-702.[CrossRef][Medline]
Nguyen, B. P., Maureen, C. R., Susana, G. G. and Carter, W. G. (2000). Deposition of laminin 5 in epidermal wounds regulates integrin signaling and adhesion. Curr. Opin. Cell Biol. 12, 554-562.[CrossRef][Medline]
Nicosia, R. F. and Madri, J. A. (1987). The microvascular extracellular matrix. Developmental changes during angiogenesis in the aortic ring-plasma clot model. Am. J. Pathol. 128, 78-90.[Abstract]
Nicosia, R. F. and Ottinetti, A. (1990). Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro. Lab. Invest. 63, 115-122.[Medline]
Niessen, C. M., Cremona, O., Daams, H., Ferraresi, S.,
Sonnenberg, A. and Marchisio, P. C. (1994). Expression of the
integrin 6ß4 in peripheral nerves: localization in Schwann and
perineural cells and different variants of the ß4 subunit. J.
Cell Sci. 107,
543-552.
Pinter, E., Barreuther, M., Lu, T., Imhof, B. and Madri, J. (1997). Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) tyrosine phosphorylation state changes during vasculogenesis in the murine conceptus. Am. J. Pathol. 150, 1523-1530.[Abstract]
Previtali, S. C., Feltri, M. L., Archelos, J. J., Quattrini, A., Wrabetz, L. and Hartung, H. (2001). Role of integrins in the peripheral nervous system. Prog. Neurobiol. 64, 35-49.[CrossRef][Medline]
Pulkkinen, L. and Uitto, J. (1999). Mutation analysis and molecular genetics of epidermolysis bullosa. Matrix Biol. 18, 29-42.[CrossRef][Medline]
Reynolds, L. E., Wyder, L., Lively, J. C., Taverna, D., Robinson, S. D., Huang, X., Sheppard, D., Hynes, R. O. and Hodivala-Dilke, K. M. (2002). Enhanced pathological angiogenesis in mice lacking ß3 integrin or ß3 and ß5 integrins. Nat. Med. 8, 27-34.[CrossRef][Medline]
Rice, F. L., Fundin, B. T., Arvidsson, J., Aldskogius, H. and Johansson, O. (1997). Comprehensive immunofluorescence and lectin binding analysis of vibrissal follicle sinus complex innervation in the mystacial pad of the rat. J. Comp. Neurol. 385, 149-184.[CrossRef][Medline]
Ryynanen, J., Jaakkola, S., Engvall, E., Peltonen, J. and Uitto, J. (1991). Expression of ß4 integrins in human skin: comparison of epidermal distribution with ß1-integrin epitopes, and modulation by calcium and vitamin D3 in cultured keratinocytes. J. Invest. Dermatol. 97, 562-567.[Abstract]
Senger, D. R. (1996). Molecular framework for angiogenesis: a complex web of interactions between extravasated plasma proteins and endothelial cell proteins induced by angiogenic cytokines. Am. J. Pathol. 149, 1-7.[Medline]
Senger, D. R., Claffey, K. P., Benes, J. E., Perruzzi, C. A.,
Sergiou, A. P. and Detmar, M. (1997). Angiogenesis promoted
by vascular endothelial growth factor: regulation through 1ß1 and
2ß1 integrins. Proc. Natl. Acad. Sci. USA
94,
13612-13617.
Senger, D. R., Perruzzi, C. A., Streit, M., Koteliansky, V. E.,
de Fougerolles, A. R. and Detmar, M. (2002). The
1ß1 and
2ß1 integrins provide critical support for
vascular endothelial growth factor signaling, endothelial cell migration, and
tumor angiogenesis. Am. J. Pathol.
160,
195-204.
Shanker, G., Olson, D., Bone, R. and Sawhney, R. (1999). Regulation of extracellular matrix proteins by transforming growth factor ß1 in cultured pulmonary endothelial cells. Cell Biol. Int. 23, 61-72.[CrossRef][Medline]
Slomp, J., Gittenberger-deGroot, A. C., van Munsteren, J. C., Huysmans, H. A., van Bockel, J. H., van Hinsbergh, V. W. and Poelmann, R. E. (1996). Nature and origin of the neointima in whole vessel wall organ culture of the human saphenous vein. Virchows Arch. 428, 59-67.[Medline]
St Croix, B., Rago, C., Velculescu, V., Traverso, G., Romans, K.
E., Montgomery, E., Lal, A., Riggins, G. J., Lengauer, C., Vogelstein, B. and
Kinzler, K. W. (2000). Genes expressed in human tumor
endothelium. Science
289,
1197-1202.
Stupack, D. G., Puente, X. S., Boutsaboualoy, S., Storgard, C.
M. and Cheresh, D. A. (2001). Apoptosis of adherent cells by
recruitment of caspase-8 to unligated integrins. J. Cell
Biol. 155,
459-470.
Trusolino, L., Bertotti, A. and Comoglio, P. M.
(2001). A signaling adapter function for 6ß4 integrin
in the control of HGF-dependent invasive growth. Cell
107,
643-654.[Medline]
van der Neut, R., Krimpenfort, P., Calafat, J., Niessen, C. M. and Sonnenberg, A. (1996). Epithelial detachment due to absence of hemidesmosomes in integrin ß4 null mice. Nat. Genet. 13, 366-369.[Medline]
Yang, J. T., Rayburn, H. and Hynes, R. O.
(1993). Embryonic mesodermal defects in 5
integrin-deficient mice. Development
119,
1093-1105.
Zimmer, D. B., Cornwall, E. H., Landar, A. and Song, W. (1995). The S100 protein family: history, function, and expression. Brain Res. Bull. 37, 417-429.[CrossRef][Medline]