© 1999 American Heart Association, Inc.
Original Contribution |
Identification of Endothelial Cell Binding Sites on the Laminin 
1 Chain
From the Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Md.
Correspondence to Hynda K. Kleinman, PhD, CDBRB, NIDR, NIH, Bldg 30, Rm 433, Bethesda, MD 20892. E-mail kleinman{at}yoda.nidr.nih.gov
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Abstract |
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Abstract—The laminins belong to a family of trimeric basement membrane glycoproteins with multiple domains, structures, and functions. Endothelial cells bind laminin-1 and form capillary-like structures when plated on a laminin-1–rich basement membrane matrix, Matrigel. Laminin-1 is composed of 3 chains,
1, ß1, and 
1. Because laminin-1 is known to contain multiple
biologically active sites, we have screened 156 synthetic overlapping
peptides spanning the entire laminin 1 chain for potential
angiogenic sequences. Only 7 of these peptides, designated as C16, C25,
C30, C38, C64, C75, and C102, disrupted the formation of capillary-like
structures by human umbilical vein endothelial cells on
Matrigel. Dose-response experiments in the presence of 50 to 200
µg/mL showed that tube formation was prevented by most peptides at
150 and 200 µg/mL, except for C16, which showed strong activity at
all concentrations. Active peptides promoted vessel sprouting from
aorta rings and angiogenesis in the chick chorioallantoic membrane
assay. In addition, the active peptides also promoted
endothelial cell adhesion to dishes coated with 0.1
µg of peptide and inhibited attachment to laminin-1 but not to
plastic or fibronectin. Four of the active peptides, C25, C38, C75, and
C102, may have cell-type specificity with endothelial
cells, since they did not promote PC12 neurite outgrowth or adhesion of
B16-F10 melanoma and human submandibular gland cells. These results
suggest that specific laminin 1–chain peptides have angiogenic
activity with potential therapeutic applications.
Key Words: angiogenesis • laminin • endothelium • extracellular matrix • adhesion
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The laminins are large basement membrane matrix glycoproteins with multiple domains, structures, and functions.1 2 Eleven different laminins have been characterized to date, and each isoform consists of 3 different chains (
, ß, and ), which form a cruciform-like
structure.3 Five , 3 ß, and 2 chains have been
identified. Laminin-1, composed of 1, ß1, and 1 chains, was
isolated from the Engelbreth-Holm-Swarm (EHS) tumor4 5 and
is the best characterized laminin.
In blood vessels, endothelial cells contact a basement
membrane that contains laminin. Although the exact nature of the
laminin isoforms present in this basement membrane has not yet been
determined, polyclonal antibodies to laminin-1 bind to this matrix,
suggesting that at least 1 of the 1, ß1, or 1 chains is
present. In vitro, endothelial cells bind to
laminin-1 and, when plated on Matrigel (a laminin-1-rich
basement membrane matrix), differentiate and form capillary-like
structures.6 It has previously been shown that laminin-1
peptides containing the IKVAV (from the 1 chain) and YIGSR (from the
ß1 chain) sequences alter the formation of capillary-like structures
on Matrigel.7 In vivo angiogenesis assays in mice have
demonstrated that the IKVAV peptide induces blood vessel formation,
whereas the YIGSR peptide has an inhibitory
effect.8 9
Recently the entire 1 chain has been duplicated by mostly 12-mer
overlapping synthetic peptides, and the biological activity of these
peptides was tested for cell adhesion with 2 tumor cell
lines.10 Several cell attachment sites were identified for
B16-F10 melanoma and HT-1080 fibrosarcoma cells. Because laminin-1 has
multiple active sites with different cell types11 12 13 and
little is known about the effect of laminin peptides on
endothelial cells, we have used an in vitro Matrigel
tube forming assay6 to identify sequences that affect in
vitro morphological differentiation. In this study, we have screened
154 overlapping synthetic peptides that span the entire laminin 1
chain, which is present in all of the laminins described to date
except laminin-5. We have identified 7 peptides (C16, C25, C30, C38,
C64, C75, and C102) that alter the formation of capillary-like
structures by endothelial cells. Aortic explant studies
showed that all 7 peptides can induce endothelial cell
sprouting, and 4 were tested in the chick choriollantoic membrane assay
and found to be highly angiogenic. Furthermore, we have shown that the
7 peptides promote endothelial cell attachment and
compete for adhesion to laminin-1. Although 3 of these peptides, C16,
C30, and C64, had been previously shown to promote tumor, neuronal, and
submandibular gland cell adhesion in vitro,10
peptides C25, C38, C75, and C102 showed cell-type specificity
with endothelial cells. These latter 4 peptides did not
promote PC12 neurite outgrowth, adhesion of B16-F10 melanoma, and human
submandibular gland (HSG) cells or inhibit HSG cell acinar formation.
Our results demonstrate that specific laminin 1 chain domains may be
important in promoting the formation of blood vessels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Matrigel and Peptides
Matrigel and laminin-1 were prepared from the EHS tumor as described.5 14 One hundred and sixty-five overlapping peptides from the
1 chain sequence15 were manually
synthesized as described by Nomizu et al.10 16 Eleven of
these peptides were insoluble and therefore could not be
tested.10
Cells and Culture
Human umbilical vein endothelial cells (HUVECs)
were obtained from freshly delivered umbilical cords by treatment with
0.1% collagenase.17 Cells were grown in RPMI
1640 containing 20% defined and supplemented bovine calf serum (BCS)
(HyClone Laboratories, Inc); 5 U/mL of heparin (Fisher Scientific); 100
U/mL penicillin; and (in µg/mL) endothelial cell
growth supplement (ECGS) 200 (Collaborative Research),
streptomycin 100, gentamycin 50, and amphotericin B 2.5 (Life
Technologies). Only early cell passages3 4 5 were used.
Acinar formation by HSG cells18 and neurite outgrowth by
PC12 cells10 were performed as previously described.
Tube Forming Assays
Screening of active peptides from the laminin 1 chain was
performed in triplicate on 96-well plates that had been coated with 106
µL of Matrigel per well. Confluent HUVECs were detached from the
plates with 0.05% trypsin and 0.53 mmol/L EDTA in HBSS (Life
Technologies). Cells were plated at a density of 13 000 cells per well
in 100 µL of HUVEC medium containing a reduced amount of BCS (10%)
and the test peptide at a concentration of 200 µg/mL. For
dose-response studies, tube assays were done using 48-well plates
containing 200 µL of Matrigel, 24 000 HUVECs, and 50 to 200 µg/mL
of test peptides. As positive controls, cells were plated in the
presence of 100 µg/mL of a peptide containing the IKVAV sequence or
HUVEC medium with only 5% BCS. The plates were incubated overnight at
37°C with 95% air/5% CO2. Cells were then
fixed and stained with Diff-Quick fixative (methanol) and solution
II (0.625 g/L each of azure A and methylene blue) (Baxter
Scientific Products). Each peptide was tested in triplicate, and
each assay was repeated at least 3 times. Tubes were scored by a
blinded observer.
Aortic Ring Sprouting Assays
Aortas were harvested from 6-week-old Sprague-Dawley rats and
cleaned of fatty tissue.19 The aorta was cross-sectioned
into thin rings with a scalpel. The rings were placed on 150 µL of
gelled Matrigel on 48-well dishes; overlaid with 50 µL of Matrigel,
which was allowed to gel for 30 minutes; and then incubated in the
presence of 100, 200, or 400 µg/mL of test peptide in 200 µL of
human endothelial serum-free medium (Life
Technologies). On the 4th day, an additional 20 µg of peptide in 100
µL of fresh serum-free medium was added, and the assay was stopped on
the 5th or 6th day after sprouts had developed. Assays were repeated 3
times in quadruplicate and scored by a blinded observer.
Cell Adhesion Assays
Adhesion of endothelial cells to laminin-1 and
to synthetic laminin 1-chain peptides was assayed on 96-well plates.
Wells were coated overnight at room temperature with either 0.1 µg of
peptide or 0.5 µg of laminin-1 in 100 µL of PBS. After decanting
unbound material, wells were rinsed 3 times with PBS, blocked for 2
hours at room temperature with 0.2% BSA in PBS, and then rinsed 3
times with PBS. Uncoated wells blocked with BSA served as controls.
Confluent cells were removed from dishes using Versene (0.2 g
EDTA/L in PBS) (Life Technologies), and 35 000
endothelial cells were plated per well in 100 µL of
RPMI 1640. After incubation for 1 hour at 37°C, plates were gently
washed twice with PBS, fixed, and stained with 20% methanol containing
0.2% crystal violet. Wells were washed with distilled
H2O until the wash was dye-free. Bound dye was
solubilized with 2% SDS and quantified in an ELISA Titertek microplate
reader at 595 nm. For inhibition assays, the cells were allowed to
attach to 96-well plates coated with either 0.5 µg of laminin-1,
fibronectin, or collagen type I in the presence of 25 to 200 µg/mL of
soluble laminin-1 peptides or as otherwise indicated. Under these
conditions sufficient peptide and protein binds.16 All
assays were performed at least 3 times in triplicate.
Chick Chorioallantoic Membrane (CAM) Assay
Approximately 4 mL of ovalbumin were removed and windows
were open from 3-day old embryonated eggs (Truslow Farms, Charlestown,
Md). On day 10, 5 µL of distilled water containing various amounts of
peptides, as specified, were dried on quartered 13-mm-diameter plastic
coverslips (Thermanox, Nalge NUNC International) and placed on the CAM
of the chick. The assay was scored and photographed on embryonal day
13. A positive score for angiogenesis was made when vessels appeared to
radiate from the peptide source.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Laminin
1–Chain Peptides Alter Endothelial Cell
Tube FormationA total of 154 synthetic peptides were screened using the Matrigel tube forming assay to identify laminin
1–chain sequences
involved in angiogenesis. Only 7 of these peptides, C16, C25, C30, C38,
C64, C75, and C102, altered tube formation at a concentration of 200
µg/mL (Table 1
). Figure 1 shows microphotographs of tube-like
structures formed by endothelial cells on Matrigel in
the absence (Figure 1A) or presence (Figure 1B through
1F) of active laminin peptides. Different peptides had varying effects
on tube morphology (Figure 1). C16 (Figure 1B) caused the
cells to form clumps, whereas the remaining peptides produced varying
degrees of incomplete tube formation. Although the reasons for the
differences in tube morphology induced by the peptides are not known,
the differences suggest that the peptides might be acting
through different mechanisms, receptors, or both. Experiments in which
the peptides were removed after 18 hours and the cells were washed and
incubated with fresh medium alone suggested that the effect of the
peptides on tube formation is irreversible, because complete
tube structures were not formed even 48 hours after peptide removal
(data not shown). Furthermore, peptide addition after tube formation
had occurred (18 hours) did not cause any apparent morphological
changes (data not shown), indicating that the peptides are not toxic
and that their presence is required during cell differentiation to be
effective. Cells remained viable after 48 hours of incubation in the
presence of peptides as determined by trypan blue exclusion, further
indicating that the effects were not due to toxicity. In an attempt to
determine the minimum concentration at which each peptide affects tube
formation, dose-response assays were performed. Most peptides were
active at the 2 highest concentrations tested, 150 and 200 µg/mL;
however, C16 showed strong activity even at 50 µg/mL (Table 1). It is important to note that the morphological
characteristics of the tubes formed at the highest doses were also
observed at lower doses, suggesting that the different morphologies
were not due to peptide concentration but rather to specific biological
effects.
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Peptides Promote Endothelial Cell Sprouting in
Aorta Rings
Because the Matrigel tube assay cannot distinguish between
angiogenic and antiangiogenic stimuli, active peptides were tested
using aorta explants. Figures 2C
through 2J show microphotographs of aorta rings in the presence of 200
µg/mL of peptides C15, C16, C64S, C64, C25, C30, C38, and C75,
respectively. All 7 active peptides (C16, C25, C30, C38, C75, and C102
[not shown]) promoted endothelial cell sprouting in a
dose-dependent manner (except C16, which was active at all doses
tested) (Table 1
) above the negative controls, which contained
either medium alone (Figure 2A) or peptides that did not disrupt
tube formation. These latter peptides included C15 (Figure 2C);
C3, C28, C57, and C63 (not shown); and a scrambled sequence of C64,
designated C64S (Figure 2E). None of the 7 peptides was
as active as the positive control, ECGS, which is a potent angiogenic
mixture containing basic and acidic fibroblast growth factors (Figure 2B). Although some of the peptides (C25, C30, C64, and C75)
appeared to promote more extensive endothelial cell
sprouting than others (C16, C38, C102), it was interesting to note that
the number and length of the sprouts also varied. For example, C75
(Figure 2J) did not promote a thick growth of cells, but the
distance the cells migrated from the tissue exceeded that observed with
the positive control. On the other hand, C16 and C25 (Figure 2D and 2G, respectively) induced strong sprouting but relatively little
migration. We conclude that all 7 active peptides have the potential to
be angiogenic in vivo because of their ability to promote microvascular
growth and migration in 3-dimensional cultures of rat aorta
explants.
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Active Peptides Promote Cell Adhesion and Inhibit Laminin-1
Binding
Peptide-coated 96-well plates were used to determine whether
peptides that altered endothelial cell tube formation
could promote cell adhesion. Endothelial cells adhered
only to those wells coated with 0.1 µg of the identified active
peptides (Table 2) with the remaining
peptides showing no activity. Inhibition experiments were performed to
determine whether various concentrations of peptides in solution could
compete for attachment to native laminin-1. The results demonstrated
that the 7 peptides (C25, C75, and C102; not shown) could prevent cell
attachment to wells coated with 0.5 µg of laminin-1 in a
dose-dependent manner (Figure 3).
Once again, C16 showed the strongest activity, since it inhibited
adhesion by 91% at 50 µg/mL, whereas the other peptides inhibited at
25% to 62%. C57, an inactive peptide in tube formation and aortic
sprouting, did not significantly inhibit adhesion at any of the
concentrations tested. These results suggest that all 7 peptides are
active binding sites in the intact laminin-1 molecule. When similar
inhibition experiments were performed on collagen type I, C16, C30, and
C38 inhibited adhesion by 46% to 88% at 200 µg/mL. When plastic or
fibronectin was used as a substrate, the peptides, with the exception
of C16, did not inhibit binding, which suggests that at least some of
the peptides are specific for laminin-1 (Table 3).
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0.025 for all peptides at the
highest dose compared with their C57 control.
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Peptides C25, C38, C75, and C102 Lack Activity With HSG, B16F10,
and PC12 Cells
Because other laminin-1 peptides, such as IKVAV and YIGSR (from
the 1 and ß1 chains, respectively), have been shown to possess
multiple biological activities with different cell types, we sought to
determine whether any of the active 1-chain peptides could promote
the adhesion of nonendothelial cells, including B16-F10
melanoma, HSG cells, and PC12 neuronal cells, and the differentiation
of the latter 2 cell lines. All 3 cell types attached to C16 and C64,
whereas C30 promoted the attachment of HUVECs and HSG cells (Table 2). C16 inhibited HSG acinar differentiation and, along with
C30, stimulated neurite outgrowth of PC12 cells (Table 2).10 In contrast, we found that the B16-F10
melanoma, HSG, and PC12 cells did not attach to C25, C38, C75, and
C102. In additional assays, these peptides did not promote PC12 neurite
outgrowth. These results suggest that these 4 peptides may be
endothelial cell specific.
Angiogenesis Is Induced In Vivo
Four peptides, C25, C30, C38, and C64, were further tested on the
chick CAM assay to determine their ability to induce angiogenesis in
vivo. As shown in Figure 4, all 4
peptides were active in inducing vessel formation in a typical radial
formation as compared with the 3 types of controls used, including
distilled water, C15 (which had been inactive in the tube, aortic
sprouting, and adhesion assays), and C64S. Figure 4A shows the
angiogenic effect of C25, C30, and C64 (panels 1 through 3) as compared
with the C64S control (panel 4). The response of the 4 active peptides
varied between 45% and 77%, with C25 being the weakest and C64 the
strongest. The controls showed a response of 20% or less (Figure 4B). These results indicate that the laminin 1 peptides that
have been identified in this study are angiogenic in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The angiogenic process consists of several steps that include degradation of the basement membrane by endothelial cells, migration to the extravascular space, proliferation, and synthesis of a new basement membrane. Because the basement membrane of the endothelium contains laminin, among other extracellular matrix proteins, and certain laminin peptides derived from the
1 and ß1 chain have been previously
shown to play a role in angiogenesis,8 20 it was important
to determine whether other sequences derived from the 1 chain affect
this process. In the present study, we have systematically screened
154 soluble peptides spanning the entire laminin 1 chain using an
assay in which endothelial cells differentiate into
organized capillary-like structures. Only 7 of these peptides disrupted
tube formation at various concentrations. Each of the 7 peptides also
promoted cell adhesion and competed for attachment to laminin-1.
Furthermore, the peptides stimulated the sprouting of
endothelial cells from aortic explants and angiogenesis
in the chick CAM assay. The peptides appear to play a role in the differentiation of endothelial cells into capillary-like structures. Removal of the active soluble peptides from the disrupted tubes did not permit the formation of complete capillary-like structures, although we cannot rule out the possibility that some peptide remained deposited on the Matrigel substrate. Furthermore, cell attachment to Matrigel was not impaired, perhaps because the cells bound to other extracellular matrix components present in Matrigel such as proteoglycans and collagen type IV. These observations, together with the fact that the peptides did not have any effect on the morphology of tubes once they formed, suggest that the peptides play a role during the differentiation of the cells. Although it is not yet known how the peptides act, the morphological differences displayed by the tubes in the presence of each peptide indicate that they might be working through different mechanisms.
Of the 7 active peptides, the following 4 may be
endothelial cell type-specific: C25, C38, C75, and
C102. The remaining 3 peptides, C16, C30, and C64, promoted the
adhesion of nonendothelial cells,10
altered endothelial cell tube formation, and promoted
the sprouting of vessels from aortic explants. C16 and C30 promoted
neurite outgrowth by PC12 cells, whereas C16 was the only peptide that
inhibited HSG acinar formation. The remaining 4 peptides identified in
our screening were only active with endothelial cells,
thus suggesting cell type specificity. Peptides C25, C38, C75, and C102
have not been previously reported to possess any type of biological
activity when tested in full peptide screens for adhesion or
differentiation of various nonendothelial cell
lines.10 These peptides, however, disrupted tube
formation, promoted endothelial cell adhesion, and
induced angiogenesis from vascular explants. Such cell-type specificity
for endothelial cells is not observed with peptides
from the 1 and ß1 chains.22 More than 679 peptides
from all 3 laminin-1 chains have been screened now in our laboratory
with endothelial cells as well as with other cell
types, and only these 4 1 chain peptides are active with
endothelial cells and not with either HSG, B16F10, or
PC12 cells. Some cell-type specificity with 1 G-domain peptides has
been previously observed with neuronal cells.13
It is not yet clear whether the 7 peptides active with
endothelial cells are functional in the intact
laminin-1 molecule in vivo. In vitro, the peptides block
endothelial cell attachment to laminin-1, and 6 of them
do not block binding to either plastic or fibronectin, although 3 of
them partially inhibited binding to collagen type I. This suggests that
most of the peptides promote cell adhesion to intact laminin-1 with
high specificity and that they have the potential to function as
attachment sites in vivo. The exact conformation of laminin-1 in vivo
is not known because of multiple interactions with itself, collagen IV,
perlecan, and other matrix components.1 Recently,
metalloproteinase 2 has been found to reveal active sites on the 2
chain of laminin-5 that were not exhibited by the intact
molecule.21 Given that intact laminin-1 does not promote
angiogenesis but the peptides do, a similar mechanism could occur with
the 1 chain in which the sequences described here could become
active as angiogenic agents during development and/or tissue
remodeling.
The active peptides are localized in different domains of the
1 chain. Only 1 of the active peptides, C102, is located on the long
arm of this chain (domain II), whereas the other 6 peptides are on the
short arm (Figure 5). Of these, C16, C25,
and C30 are in the first globular domain (domain VI), and C64 is in the
second globular domain (domain IV), suggesting that the globular
domains might be important functional sites. Peptides C38 and C75 are
present in 2 of the EGF-like repeats of domains V and III,
respectively. None of the active peptides are located in the region to
which nidogen/entactin binds (peptides 82 to 85).23 24
When a sequence comparison of all 7 peptides was performed, it was
found that 5 (C16, C25, C38 C75, and C102) shared 100% homology with
the human laminin 1 sequence and that the other 2 (C30 and C64) only
varied by 1 amino acid (Table 4).
Interestingly, these sequences are exclusively found in the laminin
1 chain and not in any other cloned proteins or laminin chains. The
1 chain is present in all known laminins, except for laminin-5,
which has a 2 chain; this suggests that the 1 chain and,
therefore, its highly conserved active sites, are important.
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Acknowledgments |
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This work was supported by intramural research funding from the National Institutes of Health.
Received August 20, 1998; accepted January 11, 1999.
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References |
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 R. Hallmann, N. Horn, M. Selg, O. Wendler, F. Pausch, and L. M. Sorokin Expression and Function of Laminins in the Embryonic and Mature Vasculature Physiol Rev, July 1, 2005; 85(3): 979 - 1000. [Abstract] [Full Text] [PDF]
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S. Hibino, M. Shibuya, J. A. Engbring, M. Mochizuki, M. Nomizu, and H. K. Kleinman Identification of an Active Site on the Laminin {alpha}5 Chain Globular Domain That Binds to CD44 and Inhibits Malignancy Cancer Res., July 15, 2004; 64(14): 4810 - 4816. [Abstract] [Full Text] [PDF]
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 E. H. Sage, M. Reed, S. E. Funk, T. Truong, M. Steadele, P. Puolakkainen, D. H. Maurice, and J. A. Bassuk Cleavage of the Matricellular Protein SPARC by Matrix Metalloproteinase 3 Produces Polypeptides That Influence Angiogenesis J. Biol. Chem., September 26, 2003; 278(39): 37849 - 37857. [Abstract] [Full Text] [PDF]
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M. L. Ponce, S. Hibino, A. M. Lebioda, M. Mochizuki, M. Nomizu, and H. K. Kleinman Identification of a Potent Peptide Antagonist to an Active Laminin-1 Sequence That Blocks Angiogenesis and Tumor Growth Cancer Res., August 15, 2003; 63(16): 5060 - 5064. [Abstract] [Full Text] [PDF]
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 Y.-J. Lee, N. Nagai, C.-H. Siar, K. Nakano, H. Nagatsuka, H. Tsujigiwa, C.-H. Roan, and M. Gunduz Angioarchitecture of Primary Oral Malignant Melanomas J. Histochem. Cytochem., November 1, 2002; 50(11): 1555 - 1562. [Abstract] [Full Text] [PDF]
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I. Okazaki, N. Suzuki, N. Nishi, A. Utani, H. Matsuura, H. Shinkai, H. Yamashita, Y. Kitagawa, and M. Nomizu Identification of Biologically Active Sequences in the Laminin alpha 4 Chain G Domain J. Biol. Chem., September 27, 2002; 277(40): 37070 - 37078. [Abstract] [Full Text] [PDF]
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 M. L. PONCE, M. NOMIZU, and H. K. KLEINMAN An angiogenic laminin site and its antagonist bind through the {alpha}v{beta}3 and {alpha}5{beta}1 integrins FASEB J, June 1, 2001; 15(8): 1389 - 1397. [Abstract] [Full Text] [PDF]
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 A. K. Sood, E. A. Seftor, M. S. Fletcher, L. M. G. Gardner, P. M. Heidger, R. E. Buller, R. E. B. Seftor, and M. J. C. Hendrix Molecular Determinants of Ovarian Cancer Plasticity Am. J. Pathol., April 1, 2001; 158(4): 1279 - 1288. [Abstract] [Full Text] [PDF]
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