1 Division of Pulmonary, Allergy and Critical Care, Department of Medicine, University of Pennsylvania, Philadelphia 19104; and 2 Centocor, Malvern, Pennsylvania 19355; and 3 University of Crete, Heraklion, GR-71409 Crete, Greece
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ABSTRACT |
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Platelet endothelial cell adhesion molecule (PECAM)-1 has been implicated in angiogenesis, but a number of issues remain unsettled, including the independent involvement of human PECAM-1 (huPECAM-1) in tumor angiogenesis and the mechanisms of its participation in vessel formation. We report for tumors grown in human skin transplanted on severe combined immunodeficiency mice that antibodies against huPECAM-1 (without simultaneous treatment with anti-VE-cadherin antibody) decreased the density of human, but not murine, vessels associated with the tumors. Anti-huPECAM-1 antibody also inhibited tube formation by human umbilical vein endothelial cells (HUVEC) and the migration of HUVEC through Matrigel-coated filters or during the repair of wounded cell monolayers. The involvement of huPECAM-1 in these processes was confirmed by the finding that expression of huPECAM-1 in cellular transfectants induced tube formation and enhanced cell motility. These data provide evidence of a role for PECAM-1 in human tumor angiogenesis (independent of VE-cadherin) and suggest that during angiogenesis PECAM-1 participates in adhesive and/or signaling phenomena required for the motility of endothelial cells and/or their subsequent organization into vascular tubes.
endothelial cells; platelet endothelial cell adhesion molecule-1; cell adhesion molecules
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INTRODUCTION |
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PLATELET
ENDOTHELIAL CELL ADHESION MOLECULE (PECAM-1) is a 130-kDa
transmembrane glycoprotein member of the Ig superfamily that is
expressed on platelets and leukocytes, as well as on endothelial cells
(EC), where it is enriched at intercellular junctions (17, 39). It was originally recognized as a protein capable of
mediating cell-cell adhesion through binding interactions with itself
or other non-PECAM-1 molecules (reviewed in Ref. 17).
Included among the list of reported PECAM-1 heterophilic ligands are
heparin-containing proteoglycans (15, 47, 65),
v
3-integrin (9, 12, 45), and
CD38 (an adenosine diphosphate-ribosyl cyclase) (14),
although the in vivo existence and/or significance of these
interactions has been questioned (6, 56, 59).
Recently, however, there is increasing evidence that, in addition to
being an adhesive protein, PECAM-1 may also participate in
intracellular signaling cascades (40). The cytoplasmic
tail of PECAM-1 contains two tyrosine residues (Y663 and Y686) that fall within a conserved signaling sequence known as the immunoreceptor tyrosine-based inhibitory motif (ITIM), a motif that was originally described in receptors that regulate immune function (63).
Phosphorylation of these two tyrosine residues in PECAM-1 through the
action of protein tyrosine kinases (e.g., Src and Csk family kinases)
(10, 33) creates docking sites for the binding and
activation of several cytosolic signaling molecules containing Src
homology 2 (SH2) domains. Included among the SH2-containing molecules
that associate with PECAM-1 are the protein tyrosine phosphatases SHP-1 and SHP-2 (10, 18, 24, 28, 29, 49), the inositol
5'-phosphatase SHIP (49), and phospholipase C-
(49). There is also evidence that PECAM-1 may associate
with phosphoinositide 3-kinase (43) and
- or
-catenin (25, 26). The ability of PECAM-1 to bind to
these various cytosolic molecules enables it to potentially modulate
the activity of intracellular signaling pathways.
PECAM-1 has been implicated in a number of important biological processes, including vascular development (2, 46), leukocyte emigration at sites of inflammation (7, 38, 60, 61), T cell activation (41, 48, 67), platelet aggregation and homeostasis (34, 42, 52), and the maintenance of vascular endothelial barrier function (21). There is also evidence that PECAM-1 plays a role in angiogenesis (16, 36, 69). These studies have demonstrated that treatment with blocking antiPECAM-1 antibodies inhibits cytokine- and tumor-induced angiogenesis in various animal models (16, 69). There is evidence, however, that angiogenesis occurring in wounded human skin grafts is inhibited only when antibodies against human PECAM-1 (huPECAM-1) and VE-cadherin are administered together, suggesting that VE-cadherin may be able to compensate for the loss of functional PECAM-1 (36). Whether this is also true for human angiogenesis occurring in other settings, such as tumor growth, is not known and is a focus of this study.
In terms of EC, angiogenesis can be viewed as a process in which these cells sever their initial cell-cell contacts, proliferate, and migrate into the perivascular matrix where they reestablish their cell-cell associations to form new patent vascular channels (11). Although the evidence does not support a role for PECAM-1 in EC proliferation (69), a number of reports have implicated PECAM-1 in EC motility (30, 53, 66) and in the endothelial cell-cell associations required for the organization of EC into tubular networks (1, 16, 22, 36, 54, 66, 69). These studies, however, have yielded conflicting results and/or have not specifically investigated the involvement of PECAM-1 in mediating EC motility separate from its putative role in promoting the formation of endothelial intercellular junctions. Consequently, the mechanisms of PECAM-1's participation in angiogenesis are still unsettled and constitute the second focus of this investigation.
Therefore, to determine the independent involvement of huPECAM-1 in human angiogenesis in a setting other than wounding, we investigated the effects of function-blocking antibodies in an in vivo murine model of the human vasculature. In this study, we show for tumors grown in human skin transplants on severe combined immunodeficiency (SCID) mice that antibodies against huPECAM-1 (without simultaneous treatment with anti-VE-cadherin antibody) decreased the density of human, but not murine, vessels associated with the tumors. These in vivo studies of huPECAM-1 were complemented by studies of in vitro tube formation and migration involving EC or cellular transfectants expressing huPECAM-1 to further investigate its mechanisms of involvement in vessel formation. Anti-huPECAM-1 antibody inhibited tube formation by human umbilical vein endothelial cells (HUVEC), as well as the migration of HUVEC through Matrigel-coated filters or during the repair of wounded cell monolayers. The involvement of huPECAM-1 in these processes was confirmed by finding that the expression of huPECAM-1 in the REN cell mesothelioma line (57) induced tube formation and enhanced cell motility. These data provide evidence of a role for PECAM-1 (independent of VE-cadherin) in human tumor angiogenesis and suggest that PECAM-1, during the formation of new vessels, may participate in adhesive and/or signaling phenomena required for the motility of EC and/or their subsequent organization into vascular tubes.
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MATERIALS AND METHODS |
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Reagents and chemicals. All reagents and chemicals were obtained from Sigma (St. Louis, MO) unless otherwise specified.
Antibodies. The following antibodies were employed: monoclonal antibodies (MAbs) 37 and 62, murine antibodies against the first Ig-like domain of huPECAM-1 (38), used in the in vivo studies; rabbit polyclonal antibody against huPECAM-1 (16) and MAb W632, murine anti-human major histocompatibility complex (MHC)-1 antibody obtained from American Type Culture Collection (ATCC; Rockville, MD), used in the in vitro studies of tube formation and migration; and MAb 390 against murine PECAM-1 (16) and an anti-huPECAM-1 antibody obtained from Immunotec (Westbrook, ME), used for immunostaining. Fluorescence-activated cell sorting (FACS) analysis confirmed that the surface expression of MHC-1 was comparable to that of PECAM-1 on HUVEC and REN cell transfectants.
Cell lines. HUVEC (Clonetics, San Diego, CA) were cultured on tissue culture surfaces preincubated with 1% gelatin in medium 199 containing 15% fetal bovine serum (FBS), 75 µg/ml endothelial growth factor, 100 µg/ml heparin, and 1 mM glutamine. HUVEC were used between passages 2 and 6. Cells from the human mesothelioma cell line, REN cells (55), were cultured in RPMI medium supplemented with 10% FBS, penicillin/streptomycin, and 2 mM L-glutamine, whereas the REN cells transfected with human PECAM-1 (57) were cultured in the same medium with G418 (0.5 g/l; GIBCO BRL, Grand Island, NY). The A549 human non-small cell lung cancer line and the M21L human melanoma line were obtained from ATCC and were cultured in RPMI or DMEM medium, respectively, each supplemented with 10% FBS, penicillin/streptomycin, and 2 mM L-glutamine.
Adenoviral infection of REN cells. REN cells were serum-starved in 1% RPMI for 24 h and then transduced with adenovirus constructs at multiplicity of infection of 5 in 2% RPMI for 48 h at 37°C. Adenovirus constructs were a full-length PECAM-1 (ad.PECAM-1) and LacZ control (ad.LacZ). A nontransduced set of cells was treated identically but without adenovirus exposure. Cells were then trypsinized, and high PECAM-1 expression was confirmed by FACS analysis. LACZ expression was confirmed with the LACZ assay (Promega, Madison, WI).
Skin transplantation. The protocols for skin transplantation have been described previously in detail (13, 38). Briefly, SCID mice were obtained from a colony maintained at the Wistar Institute Animal Facility. At 4-6 wk of age, the plasma of each mouse was tested for IgM production, and only fully immunodeficient mice were used. Neonatal foreskins from elective circumcisions obtained by using sterile techniques were cut into two halves and trimmed to a diameter of 1-1.5 cm. Full-thickness human skin grafts from the same donor were then transplanted into full-thickness, size-matched wound beds prepared on each flank of a SCID mouse. Mice were used for experiments only within the second month following human skin transplantation to ensure engraftment and stabilization. Only those mice whose grafts grossly showed no signs of inflammation or rejection were used.
Tumor implantation and antibody injections. Tumor cells from subconfluent tissue culture flasks were removed with trypsin-EDTA and washed, and 2 × 106 M21L cells or 4 × 106 A549 cells in a total volume of 50 µl of complete medium were injected intradermally into each human skin graft. For each mouse, 200 µg of nonimmune murine IgG or antibody were administered intraperitoneally 3 times/wk for 3 wk, beginning 1 wk after tumor cell injection. The animals were killed 1 mo after the start of the experiment. For analysis of tumor growth, the mice were killed, tumor volume was measured, and the tumor with surrounding human skin was snap frozen in OCT for immunohistochemical analysis.
Immunohistochemical staining and quantitation of tumor angiogenesis. Immunohistochemistry was performed using a commercially available kit, according to the manufacturer's instructions (Vector M.O.M Immunodetection kit; Vector Laboratories, Burlingame, CA). Briefly, 6-µm-thick sections were prepared by cryostat, transferred to glass slides and fixed in ice-cold acetone, and rinsed in PBS. The sections were then permeablized with 0.3% Triton X-100 in PBS for 10 min, treated with 0.5% H202 in PBS for 10 min, and then blocked with mouse IgG blocking reagent for 1 h. The primary antibody was applied for 1 h, washed, and then incubated with biotinylated secondary antibody for 30 min. The reaction was developed with avidin-biotin complex reaction, and the sections were lightly counterstained with hematoxylin.
Identification of human vessels was accomplished by staining with an anti-human PECAM-1 antibody (Immunotec). In separate studies, we confirmed that the binding of this antibody was not competitively inhibited by MAbs 37 or 62 and that the number of human vessels labeled by this antibody was comparable to that seen when sections were stained with biotinylated Ulex europaeus agglutinin I (Vector Laboratories), a reagent that specifically recognizes human vessels. Murine vessels were identified with MAb 390, an anti-murine PECAM-1 antibody (16). For quantitation of the angiogenic response, the vessel density at the margins of the tumors was determined as follows. Serial sections were obtained from different levels within the tumor separated by ~100 µm and stained with anti-human or anti-murine PECAM-1 antibodies to identify human or murine vessels, respectively. A total of eight levels per tumor were analyzed. By using computer-assisted image analysis, an optical field of 3.6 × 105 µm2 was defined and positioned at serial locations along the margins of the tumor so that the tumor edge bisected the optical field into two equal sections. At a given position, the area of the optical field occupied by either murine or human vessels was then determined. For each section, 8-12 optical fields were analyzed.In vitro tube formation assay. In vitro tube formation was studied using previously described procedures (36, 39). Matrigel (Collaborative Biomedical Products, Bedford, MA) was diluted with cold, serum-free medium to 10 mg/ml. Next, 50 µl of the solution were added to each well of a 96-well plate and allowed to form a gel at 37°C for 30 min. EC (30,000 cells) in 200 µl of complete medium with BSA or antibody were subsequently added to each well and incubated for 10 h at 37°C in 5% CO2. Under these conditions, EC form delicate networks of tubes that are detectable within 2-3 h and are fully developed after 8-12 h. After incubation with BSA or antibody, the wells were washed and the Matrigel and its endothelial tubes were fixed with 3% paraformaldhyde. Total tube length per well was determined by computer-assisted image analysis using the Image-Pro Plus program (Media Cybernetics, Silver Spring, MD). For studies of tube formation by REN cells, it was noted that these cells formed tubes on Matrigel but not on a mixture of Matrigel and collagen. Therefore, these studies with REN cells were conducted with a Matrigel-collagen mixture that was prepared as follows: 2 volumes of Matrigel (10 mg/ml) were mixed with 1 volume of a collagen solution prepared by mixing rat tail collagen type I (Collaborative Biomedical Products; stock concentration 3-4 mg/ml), NaHCO3 (1.76 mg/ml), and 10× DMEM together in a ratio (by volume) of 7:2:1, respectively.
In vitro wounding assay. Endothelial wounding was modified from previously published procedures (23). EC (20,000 cells) were added to 24-well tissue culture plates and allowed to grow to confluence. Circular defects were then made in the monolayer by placing the tip of a glass Pasteur pipette fitted with a rubber ring onto the monolayer and then applying suction to lift the cells off the plastic. This resulted in uniform circular wounds of ~30 µm2 in area with minimal trauma to the monolayer. The wounded culture was washed with PBS and then incubated for 24 h in medium (with 1% serum) containing BSA or antibody. After incubation, the monolayers were fixed with 3% paraformaldhyde and stained with crystal violet, and the area was determined by computer-assisted image analysis using the Image-Pro Plus program (Media Cybernetics). In each experiment, an initial set of wounds (n = 3) was made and immediately fixed and stained to give a measure of the initial area. For each antibody condition, the data are expressed as the change in wound area.
In vitro Matrigel invasion/migration assay. Matrigel-coated transwell inserts (Costar; 8-µm pore filter) were prepared by twice adding 100 µl of Matrigel (250 µg/ml) to the transwell and allowing the Matrigel to dry at 37°C in a nonhumidified oven for 24 h. HUVEC or REN cells (100,000 cells) were labeled overnight with [3H]thymidine and resuspended in low-serum medium (5% serum) containing BSA or antibody. The resulting cell suspensions were then placed in transwell filter inserts, which in turn were placed in 12-well plates containing high-serum medium (10% for REN and 20% for HUVEC) and incubated for 8 h at 37°C in 5% CO2. The cells migrated through the Matrigel, passaged through the pores of the filter, and adhered on the lower surface of the filter (27). After incubation with BSA or antibody, the wells were removed and washed, and the top surface of the filter was wiped with a cotton swab. The filters were then carefully cut out, placed in scintillation fluid, and counted in a beta-counter. For each antibody condition, migration was expressed as the percentage of cells migrated.
Single-cell migration. Time-lapse video microscopy was performed as previously described (35). For these studies, slides obtained from Lab-Tek (Micro Video Instruments, Arrow, MA), which were sealed with a mixture of petroleum jelly and paraffin (20:1) to maintain the pH of the medium, were coated with fibronectin (10 µg/ml) for 1 h at 37°C and blocked with 2% BSA for 1 h at 37°C. The cells were plated on the slides at a density of 0.5 × 105 cells and then placed in a 37°C-humidified Plexiglas microscope culture chamber (Nikon). A field containing several REN cells was selected and observed under phase contrast for 7 h. Motile activity was studied by measuring the total distance covered by the migrating cells using time-lapse video microscopy. Sequential images were collected at 1-h intervals. A minimum of 70 cells was studied in each experiment for each condition.
Statistical analysis. Differences among groups were analyzed using one-way analysis of variance. Results are presented as means ± SE. When statistically significant differences were found (P < 0.05), individual comparisons were made using the Bonferroni/Dunn test.
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RESULTS |
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Antibody against huPECAM-1 inhibits tumor-associated human angiogenesis in human skin grafts. Recent studies in rodents have demonstrated that treatment with blocking anti-PECAM-1 antibodies inhibits cytokine- and tumor-induced angiogenesis in these animals (16, 69). However, it is currently unknown whether antagonism of huPECAM-1, without concomitant inhibition of VE-cadherin, might also inhibit tumor-associated human angiogenesis. To address this question, we made use of a model in which human skin (neonatal foreskin) is transplanted onto SCID mice (8, 13, 38). In this system the vasculature of the grafts consists of both murine and human vessels, with the human vessels retaining the capacity to respond to inflammatory or angiogenic stimuli (8, 13, 38).
We therefore studied the effect of anti-huPECAM-1 antibody (MAb 62) on the growth and angiogenic response of a human non-small cell lung cancer line (A549) and a human melanoma line (M21L) injected into human skin grafts on SCID mice. MAb 62 has been shown previously to be a function-blocking anti-huPECAM-1 antibody that does not cross-react with murine PECAM-1 and whose binding epitope is in the first Ig-like domain of PECAM-1 (38). For each mouse, 200 µg of nonimmune murine IgG or antibody were administered 3 times/wk for 3 wk, beginning 1 wk after tumor cell injection. The animals were killed 1 mo after the start of the experiment. Treatment with MAb 62 did not significantly inhibit tumor growth (as assessed by tumor volume) or the density of murine vessels at the periphery of the tumors (Table 1 and Fig. 1). In contrast to this, the density of human vessels found at the tumor margins was significantly reduced for both tumors (38% for the A549 tumor and 65% for the M21L tumor). Similar results were obtained with a second domain 1-specific anti-PECAM-1 antibody (MAb 37, data not shown). These data suggest that anti- PECAM-1 antibodies were able to inhibit tumor-induced human angiogenesis in this system but that there was sufficient murine angiogenesis to sustain growth of these tumors.
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In vitro tube formation by huPECAM-1-expressing cells.
The mechanisms by which anti-huPECAM-1 antibodies might block in vivo
angiogenesis are still unclear. Certainly a critical step in the
formation of new vessels is the organization of proliferating and
migrating EC into stable tubular networks. As shown in Fig. 2, we found that antibody against
huPECAM-1 (16) limited the ability of HUVEC to organize
into tubular structures on Matrigel, a substrate composed of basement
membrane proteins. These data are in agreement with previous studies in
which tube formation by rat EC on collagen and murine EC on Matrigel
were inhibited by specific anti-PECAM-1 antibodies (16,
69). Together, these studies provide strong evidence that
PECAM-1 is involved in in vitro endothelial tube formation.
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Wound-induced migration of huPECAM-1-expressing cells.
In a quiescent vessel, EC are associated with other EC, and thus EC
migration during angiogenesis must initially, and necessarily, involve
the disruption of these cell-cell associations. We therefore studied
the effect of anti-huPECAM-1 antibody (16) on the closure of circular defects made in confluent monolayers of HUVEC. In this
assay, under our experimental conditions, EC broke their cell-cell
contacts and migrated into the wound to close the defect. Compared with
antibody controls, anti-PECAM-1 antibody delayed the closure of wounds
in a dose-dependent manner (data shown for 100 µg/ml) (Fig.
4A). For HUVEC, the anti-human
antibody inhibited wound closure by 35%. Because these antibodies did
not have any effect on the proliferation of these cells (data not
shown), the delay of wound closure was most consistent with an
inhibition of EC migration. Expression of huPECAM-1 in REN cells
increased the rate of wound closure compared with that of the
nontransfected REN cells, an effect that was nearly prevented by the
inclusion of PECAM-1 antibody during the 24-h repair period (Fig.
4B). Together, these data suggest the involvement of PECAM-1
during wound-induced EC migration.
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Invasion/migration of huPECAM-1-expressing cells through Matrigel.
Although the data presented above provide evidence that expression of
PECAM-1 promotes EC migration, studies of wound-induced migration
involving molecules that mediate intercellular interactions, such as
PECAM-1, are unavoidably confounded by the presence of cell-cell
associations. Therefore, to further investigate the involvement of
PECAM-1 in EC migration independent of cell-cell interactions, we
studied the effect of anti-huPECAM-1 antibody on the ability of HUVEC
as single cells to migrate through transwell filter inserts coated with
Matrigel. This assay is particularly relevant because the early stages
of angiogenesis involve the migration of EC into the surrounding
perivascular matrix, a phenomenon that, in vivo, initially includes
invasion of and movement through the matrix of the basement membrane.
As shown in Fig. 5A,
anti-huPECAM-1 antibody (16), compared with anti-MHC-1,
inhibited the passage of HUVEC through Matrigel in a dose-dependent
manner (data shown for 100 µg/ml). These data suggest that, for EC,
PECAM-1 may be involved in their migration through proteins that make
up the basement membrane. This conclusion was supported by the finding that expression of huPECAM-1 in REN cells increased the rate of migration of these cells through Matrigel compared with that of the
nontransfected REN cells in a manner that was inhibited by anti-huPECAM-1 antibody (Fig. 5B).
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Single-cell migration of huPECAM-1-expressing cells.
To further confirm the involvement of PECAM-1 in EC migration, we
studied the single-cell random migration over 24 h of REN cells
transduced with adenoviral constructs expressing LacZ or huPECAM-1 by
using time-lapse video microscopy. We observed that expression of
huPECAM-1 by adenoviral infection increased the random motility of REN
cells (Fig. 6). Together, these data
indicate that PECAM-1 may play a role in EC migration independent of
effects on endothelial cell-cell interactions.
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DISCUSSION |
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To investigate the involvement of huPECAM-1 in angiogenesis, we studied the effects of function-blocking antibodies in an in vivo model of the human vasculature and in vitro assays of tube formation and migration involving EC and cellular transfectants expressing huPECAM-1. We observed for tumors grown in human skin transplants on SCID mice that antibodies against huPECAM-1 decreased the density of human, but not murine, vessels associated with the tumors. Anti-huPECAM-1 antibody also inhibited tube formation by HUVEC as well as the migration of HUVEC through Matrigel-coated filters or during the repair of wounded cell monolayers. The involvement of huPECAM-1 in these processes was confirmed by finding that the expression of huPECAM-1 in the REN cell mesothelioma line induced tube formation and enhanced cell motility. These data provide evidence of a role for PECAM-1 in human angiogenesis and suggest that PECAM-1 during the formation of new vessels may participate in adhesive and/or signaling phenomena required for the motility of EC and/or their subsequent organization into vascular tubes.
Evidence for the involvement of PECAM-1 during in vivo angiogenesis has come from studies showing that treatment with anti-PECAM-1 antibody inhibits vessel formation in rodent models of cytokine- or tumor-induced angiogenesis (16, 69). Our finding that antibody against huPECAM-1 also limits the vascularization by human vessels of tumors in human skin transplants (Fig. 1; Table 1) extends our previous work and implicates the independent involvement of PECAM-1 in human (tumor) angiogenesis. These data, however, do differ somewhat from the previous study by Matsumura et al. (36). In that study, it was reported that wound-induced angiogenesis in human skin grafts placed on SCID mice was inhibited only when antibodies against huPECAM-1 or VE-cadherin were given together, but not when they were administered individually. The differences between these studies may be related to antibodies used but may also suggest that VE-cadherin may substitute for PECAM-1 or that the requirement of PECAM-1 during in vivo vessel formation may depend on the angiogenic stimulus.
The mechanisms of PECAM-1's involvement in angiogenesis are still being defined. Unquestionably an important step in the formation of new vessels is the organization of proliferating and migrating EC into patent endothelium-lined channels with stable cell-cell contacts (11). The presence of PECAM-1 in intercellular junctions (51) and the previous observation that anti-PECAM-1 antibody disrupts the formation of tight monolayers by EC (1) have led to the hypothesis that PECAM-1 may regulate the assembly of junctional complexes required for the stable association of adjacent EC in the wall of the vessel (22). Our finding that EC tube formation was inhibited by anti-huPECAM-1 antibody (Fig. 2) and that expression of huPECAM-1 promoted the formation of tubular networks by cellular transfectants (Fig. 3) is consistent with this hypothesis.
Recent studies on the signaling properties of PECAM-1 have
suggested a mechanism by which PECAM-1 might regulate the
organization or stability of EC junctional structures. Its cytoplasmic
domain possess two tyrosine residues (Y663 and Y686) that fall within a
conserved signaling motif known as an ITIM (40).
Phosphorylation of these residues creates a docking site for signaling
proteins that bind via SH2 domains, such as the phosphatases SHP-1 and SHP-2 (10, 18, 24, 28, 29, 49). In addition to
SH2-containing molecules, PECAM-1 has also been recently reported to
associate with - and
-catenin and to promote their localization
to cell-cell junctions (25, 26). Of note, phosphorylation
of catenins has been reported to destabilize cadherin-dependent
cell-cell adhesion (3, 37). Given the above and the
observation that PECAM-1 is tyrosine phosphorylated in confluent EC
monolayers (20, 32), it is possible that during the
organization of EC into vascular channels, PECAM-1 at newly emerging
endothelial cell-cell contacts recruits catenins to intercellular
junctions. This association with PECAM-1 brings the catenins into close
proximity to molecules that modulate their phosphorylation state.
Specifically, net phosphatase activity associated with PECAM-1 may
provide for levels of dephosphorylated catenin that allow the formation
and/or persistence of junctional complexes required for stable
endothelial cell-cell contacts.
In interpreting the results of studies by us and others of tube formation by PECAM-1-expressing cells (16, 36, 54, 69), it is important to recognize that the assays employed involve more than just the ability of the cells to associate with each other. These assays also depend on the motility of the cells used and the ability of those cells to undergo shape changes before assembly into tubular networks. Consequently, we also investigated the potential involvement of PECAM-1 in cell migration. In studies of wound-induced migration (Fig. 4) and single-cell migration through Matrigel (Fig. 5), we found that 1) PECAM-1 antibody blocked the motility of HUVEC and 2) expression of PECAM-1 increased cell migration. In comparable studies using these assays with a murine EC line, the anti-murine PECAM-1 MAb 390 inhibited cell migration (data not shown). Together, these data suggest a role for PECAM-1 in EC motility independent of any effects it might have on endothelial cell-cell interactions, a suggestion that is further supported by the finding that expression of PECAM-1 enhances random single-cell motility of cellular transfectants (Fig. 6). These data are in agreement with the studies of HUVEC cultured in a type I collagen gel matrix by Yang et al. (30), who found that cell elongation and cell migration were inhibited in the presence of PECAM-1 antibodies.
Our data, however, differ from several earlier studies that investigated the role of PECAM-1 in EC migration (30, 52). In these reports, expression of PECAM-1 in murine fibroblasts or in the ECV304 tumor line decreased migration, an effect that depended on the presence of the extracellular domain and Y686 in the cytoplasmic domain. Of note, these studies relied exclusively on assays in which cells initially in confluent monolayers were induced to move either by wounding of the monolayer or by the removal of circular fences that prevented their outward radial movement. The challenge in interpreting these studies of "sheet migration" is that cell movement in these assays is dependent on the ability of the cells to break free from their cell-cell contacts, as well as on actual cell motility. We would further acknowledge that some of the conflict in the results may be related to differences in the cells that were employed in the transfections (mesothelioma vs. fibroblasts or ECV304) and/or the EC (bovine vs. human EC) that were studied.
Although the mechanism(s) by which PECAM-1 might promote EC motility
have not been determined, there is evidence that PECAM-1 may mediate
its effects on cell migration by modulating integrin function. Numerous
studies have demonstrated that simulation of ligand binding by the
addition of anti-PECAM-1 antibodies upregulates the function of
1- (31, 58),
2- (4, 5,
19, 44), and
3-integrins (12, 62).
More recently it has been shown that PECAM-1-dependent integrin
activation requires oligomerization of the molecule (68)
and, in leukocytes, may be mediated by Rap1, a small Ras-related GTPase
(50). Because the interactions of integrins with
extracellular matrix are critical for cell motility, ligand engagement
of PECAM-1 may stimulate the activity of integrins required for cell
migration. While admittedly still in contention (6, 56,
59), the ligand interactions that might trigger these
processes include the binding of PECAM-1 to proteoglycans in the matrix
(15, 47, 64) or interactions of PECAM-1 with
v
3-integrin on the same EC
(65) or on stromal cells.
In conclusion, our data, as well as those of others, allow us to
present an initial working model for the involvement of PECAM-1 in
angiogenesis. In quiescent vessels, PECAM-1-PECAM-1 homophilic interactions in intercellular junctions promotes the formation of
stable EC-EC associations by modulating the phosphorylation state of
catenins and possibly other molecules critical to formation of
junctional complexes. This suppresses the migratory phenotype of the
EC. However, in the context of an angiogenic stimulus, angiogenic
factors induce a redistribution of PECAM-1 out of EC junctions
(51), resulting in the loss of the inhibition of
cell migration and increased surface PECAM-1 available to participate in heterophilic interactions. This could potentially involve
interactions with v
3 on endothelial or
stromal cells or with proteoglycans in the extracellular matrix. These
ligand interactions in turn directly (if
v
3 is the ligand) or indirectly trigger
integrin-dependent phenomena such as EC migration. Later, as EC are
reorganizing into vascular tubes, the reestablishment of
PECAM-1-PECAM-1 interactions facilitates the stabilization of initial
cadherin/catenin-dependent endothelial cell-cell associations, thereby
suppressing EC motility and thus promoting the formation of vascular
tubes. Studies are now underway to test this hypothesis.
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ACKNOWLEDGEMENTS |
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The very helpful advice and support of Dr. Steven Albelda, as well as the provision of REN cell transfectants for these studies, is greatly appreciated.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-62254 (H. M. DeLisser) and HL-04248 (C. D. O'Brien).
Address for reprint requests and other correspondence: H. M. DeLisser, 806 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160 (E-mail: delisser{at}mail.med.upenn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 9, 2002;10.1152/ajpcell.00524.2001
Received 1 November 2001; accepted in final form 1 January 2002.
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