© 1995 American Heart Association, Inc.
Articles |
Endothelial Cell Interactions With Synthetic Peptides From the Carboxyl-Terminal Heparin-Binding Domains of Fibronectin
From the Department of Laboratory Medicine and Pathology/Biomedical Engineering Center (J.C.H., J.B.M., D.L.M.), University of Minnesota, Minneapolis, and the Department of Pathology (C.A.D.), Wayne State University School of Medicine, Detroit, Mich.
Correspondence to Dr Daniel L. Mooradian, University of Minnesota, Laboratory Medicine and Pathology, Box 107, Mayo Memorial Building UMHC, Minneapolis, MN 55455.
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Abstract |
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Abstract Fibronectin (FN) plays an important role in endothelial cell adhesion, spreading, and motility. Within FN, a number of functional domains have been identified, including the 33/66-kD carboxyl-terminal heparin-binding fragments, which support the adhesion of vascular endothelial cells. A number of synthetic peptides representing amino acid sequences within the 33/66-kD fragments have been shown to promote the adhesion, spreading, and migration of a variety of cell types. Our working hypothesis is that one or more of these sequences may also mediate vascular endothelial cell adhesion, spreading, and migration to the 33/66-kD fragments. In support of this hypothesis, we have demonstrated that endothelial cells from various sources adhered in a concentration-dependent manner to surfaces coated with FN, the 33/66-kD fragments, and synthetic peptides derived from the 33/66-kD fragments of FN. FN and the 33/66-kD fragments also promoted endothelial cell spreading and migration. Although each of the six synthetic peptides tested supported endothelial cell adhesion, only one of these peptides within the carboxyl-terminal heparin-binding domain (FN-C/H-V) promoted endothelial cell spreading and migration. Cell spreading on FN-C/H-V, as well as on FN and the 33/66-kD fragments, was associated with the formation of a well-developed actin cytoskeleton and the formation of focal contacts. FN-C/H-V (but not scrambled FN-C/H-V) inhibited cell spreading on FN and the 33/66-kD fragments in a concentration-dependent manner. FN-C/H-V had a modest effect on the adhesion of a clonal population of rat heart endothelial cells (RHE-1A) to the 33/66-kD fragments of FN and no effect on RHE-1A cell adhesion to FN. These findings suggest that peptide FN-C/H-V is unique among this group of peptides derived from the 33/66-kD heparin-binding fragments of FN in its ability to promote the adhesion, spreading, and migration of vascular endothelial cells and further suggest that the sequence defined by this peptide plays an important role in vascular endothelial cell interactions with the 33/66-kD fragments of FN.
Key Words: adhesion • spreading • endothelium • fibronectin • migration
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The extracellular matrix has been implicated as a regulator of endothelial cell adhesion, spreading, and motility.1 2 3 4 These biological processes play a role in the maintenance of vascular integrity, endothelial cell polarity, and vessel repair after injury. An understanding of the complex molecular interactions between the endothelium and extracellular matrix may provide an opportunity to directly influence endothelial cell behavior during angiogenesis, vascular repair, and the repopulation of synthetic vascular grafts after autologous endothelial cell seeding.
Fibronectin (FN), found in plasma and in extracellular
matrices,5 plays an important role in endothelial cell
adhesion, spreading, and motility.6 7 FN is a large
heterodimeric glycoprotein composed of similar but not identical
polypeptide subunits, termed the A and B chains, that are linked by
disulfide bonds.8 A number of functionally distinct
domains of FN have been identified (Fig 1
, Table 1), including several domains that support cell
adhesion.5 The RGDS tetrapeptide sequence is perhaps the
most widely known.9 Vascular endothelial cells adhere to a
fragment of FN containing this sequence,10 and Hayman et
al11 have shown that an RGDS-containing synthetic peptide
can inhibit vascular endothelial cell adhesion to intact FN. The RGDS
sequence mediates cell adhesion via interactions with the
5ß1 integrin.12 Recent
reports suggest that the activity of the RGDS-containing central
cell-binding domain of FN may be dependent on one or more
"synergy" sites that contribute to full activity of this
domain.13 14 15
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The type III connecting segment, a region of alternative
splicing,16 is a second cell-adhesion–promoting domain in
FN,17 and two peptides within this domain have also been
found to possess cell-adhesion–promoting activity18 : (1)
peptide CS-1, which contains a sequence (LDV) that promotes cell
type–specific adhesion,19 and (2) peptide CS-5, which is
present in cellular but not plasma FN,16 20 contains
the related sequence REDV, and also promotes cell
adhesion.18 Cell adhesion to both CS-1 and CS-5 is
mediated by the 4ß1
integrin.20 21 Several studies indicate that human
umbilical vein endothelial cells (HUVECs) do not express the
4ß1 integrin,21 22 but a
recent study by Massia and Hubbell23 suggests that HUVECs
do express the 4ß1 integrin.
The 33/66-kD carboxyl-terminal heparin-binding fragments of FN also support the adhesion of various cell types,24 including HUVECs.10 Previous studies have identified a number of peptide sequences within the carboxyl-terminal heparin-binding domains that promote cell adhesion. Two of these peptides, FN-C/H-I25 26 and FN-C/H-II,26 27 28 promote melanoma and neuroblastoma cell adhesion, respectively. Recent evidence suggests that melanoma cells may adhere to these sequences via a cell-surface heparan sulfate proteoglycan.29 In addition, a third peptide, FN-C/H-III, has recently been shown to support melanoma cell adhesion.30 Cell adhesion to this group of peptides is cell-type specific, as illustrated by the fact that peptides that support the adhesion of one cell type (eg, corneal epithelial cells)31 do not support the adhesion of other cell types (eg, human keratinocytes).27 Recently, two additional peptide sequences, FN-C/H-IV and FN-C/H-V, have been shown to promote rabbit corneal epithelial cell adhesion and spreading, and FN-C/H-V has been identified as a novel sequence that promoted haptotactic migration of this cell line.32 Furthermore, FN-C/H-V has recently been shown to support human keratinocyte adhesion and spreading.33
Our working hypothesis is that one or more of these peptide sequences may also mediate vascular endothelial cell adhesion, spreading, and migration in response to the 33/66-kD fragments. In support of this hypothesis, we have demonstrated that endothelial cells from various sources adhered in a concentration-dependent manner to surfaces coated with FN, the 33/66-kD fragments, and a number of synthetic peptides derived from the 33/66-kD fragments of FN. FN, the 33/66-kD fragments, and one of the six synthetic peptides tested (FN-C/H-V) also promoted endothelial cell spreading and migration. FN-C/H-V (but not scrambled FN-C/H-V) inhibited cell spreading on FN and the 33/66-kD fragments in a concentration-dependent manner. FN-C/H-V had a modest effect on the adhesion of a clonal population of rat heart endothelial cells (RHE-1A) to the 33/66-kD fragments of FN and no effect on RHE-1A cell adhesion to FN. These results demonstrate that peptide FN-C/H-V is unique among this group of peptides derived from the 33/66-kD fragments of FN in its ability to promote the adhesion, spreading, and motility of vascular endothelial cells and suggest that the sequence defined by this peptide plays an important role in vascular endothelial cell interactions with the 33/66-kD fragments of FN.
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Materials and Methods |
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Cell Culture
RHE-1A34 was the principal endothelial cell population used in the present study. These cells exhibited a typical endothelial cell cobblestone morphology and positive immunofluorescent staining for factor VIII–related antigen. RHE-1A cells were maintained in DMEM (basal medium) containing 10% fetal bovine serum (FBS) and gentamicin (50 µg/mL) (Sigma Chemical Co). Cell cultures were incubated at 37°C in an atmosphere of 5% CO2 in air, and the medium was changed three times weekly. Cultures were subcultured every 4 days at a split ratio of 1:5 by using 5% trypsin (Sigma) plus 0.5% EDTA (Sigma) (trypsin/EDTA). RHE-1A cells were used at passage 25 and below. Bovine aortic endothelial cells (BAECs)35 were maintained in the same manner as RHE-1A cells. These cells also exhibited a typical endothelial cell cobblestone morphology and positive immunofluorescent staining for factor VIII–related antigen. BAECs were used at passage 5 and below. HUVECs (passage 2) were obtained from Clonetics Corp and maintained in MCDB 131 (basal medium) containing 10 ng/mL epidermal growth factor, 1 µg/mL hydrocortisone, 10 µg/mL heparin, 2% fetal bovine serum, and 0.4% bovine brain extract (Clonetics Corp). Cell cultures were incubated at 37°C in an atmosphere of 5% CO2 in air, and the medium was changed every other day. Cultures were subcultured every 4 days at a split ratio of 1:5 by using trypsin/EDTA. HUVECs were used at passages 3 and 4.
Extracellular Matrix Proteins
Human plasma FN was purified as previously
described,36 and the 33/66-kD heparin-binding/cell
adhesion–promoting fragments were generated by proteolytic digestion
of intact FN and were purified as previously
described.24
Purified FN and FN fragments were subjected to SDS-PAGE according to
the method of Laemmli.37 FN migrated as a closely spaced
doublet of 230 and 240 kD under reducing conditions and was found to be
>98% pure on the basis of Coomassie blue staining. Preparations of
the 33/66-kD fragments contained two major bands with molecular masses
of 
33 and 66 kD. These preparations did not contain contaminant FN
or 75-kD fragments at levels that were detectable by Coomassie blue
staining.
Murine laminin was obtained from GIBCO BRL, Inc, and bovine type I collagen (Vitrogen) was obtained from Celtrix, Inc.
Peptide Synthesis and Characterization
Synthetic peptides were synthesized at the Microchemical
Facility (University of Minnesota) on an Applied Biosystems peptide
synthesizer by using previously described modifications of the
Merrifield solid-phase method (Koliakos et al38 ). Crude
peptides were purified by preparative reversed-phase high-performance
liquid chromatography (HPLC) on a C-18 column and were eluted with a
linear gradient of acetonitrile (0% to 60%) containing 0.1%
trifluoroacetic acid in water. Peptide composition was verified by
amino acid analysis before use. Peptide purity was >95% on the
basis of analytical HPLC. The sequences and selected properties of the
synthetic peptides used in the present study are shown in Fig 1
and
Table 1. Hydropathy indexes were calculated by using the method of Kyte
and Doolittle.39
Peptide Conjugation to Ovalbumin
Synthetic peptides used as substrates in cell adhesion
experiments and in cell motility experiments were conjugated to
ovalbumin (OVA) by using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
(Sigma) as previously described.26 The conjugation of
adhesion-promoting peptides to carrier proteins such as albumin has
been shown to enhance their adhesion-promoting activity when adsorbed
on surfaces.40 Briefly, 5 mg of each peptide was dissolved
in 1 mL of water on ice, and 5 mg of OVA was then added. EDC (50 mg) in
150 µL of water was added to the peptide-OVA mixture, and the
solution was mixed overnight at 4°C on a rotary shaker. The
OVA-conjugated peptides were then dialyzed extensively against PBS (pH
7.4) to remove excess EDC as well as any unconjugated peptide
(Spectrapore 6, 30-kD cutoff, Spectrum Medical Industries). The average
coupling efficiency was 35% and was determined by using
125I-labeled peptides of known specific activity and
measuring the radioactivity present in purified peptide-OVA
conjugates. Peptide-OVA conjugates were stored at -80°C.
Endothelial Cell Adhesion Assay
Endothelial cells were grown to 70% to 80% confluence in
75-cm2 polystyrene flasks and labeled with 10 µCi/mL
[35S]methionine (Tran 35S-label, ICN
Radiochemicals) for 24 hours. Purified proteins, OVA-conjugated
peptides, or OVA-conjugated OVA was diluted in Voller's carbonate
buffer (Na2CO3, 1.59 g;
NaHCO3, 2.93 g; and NaN3, 0.2 g
in 1 L ddH2O [pH 9.6]), and 50-µL aliquots were added
to the wells of a 96-well Immulon I microtiter plate (Dynatech
Laboratories, Inc). Proteins or peptides were allowed to adsorb
overnight at 37°C, at which time nonspecific binding sites were
blocked by using PBS containing 2 mg/mL OVA. Subconfluent endothelial
cells were harvested by using trypsin/EDTA and resuspended in basal
medium containing 10% FBS. The cells were centrifuged and resuspended
twice in basal medium containing 2 mg/mL OVA, counted, and resuspended
in basal medium containing 2 mg/mL OVA at a final cell density of
5x104 cells per milliliter. Aliquots (100 µL) of this
cell suspension were added to protein/peptide-coated wells and
incubated at 37°C. After 1 hour, nonadherent cells were removed by
washing three times with basal medium (200 µL per well). After the
cells were washed, lysis buffer (1% SDS/0.5N NaOH) was added (100
µL) to each well. After cell lysis (30 minutes at 60°C), the
content of each well was transferred to a scintillation vial containing
5 mL scintillation cocktail and 0.1 mL water, and radioactivity was
measured in a Beckman 3801 scintillation counter.
To assess the role of the sequence defined by FN-C/H-V in RHE-1A cell adhesion to FN and the 33/66-kD fragments, RHE-1A cells were preincubated for 60 minutes at 37°C with 5, 10, 50, 100, or 500 µg/mL FN-C/H-V or scrambled FN-C/H-V. These peptides were not conjugated to OVA. Cells treated in this manner were then added to Immulon I plates coated with FN (5 µg/mL), the 33/66-kD fragments of FN (5 µg/mL), or OVA-conjugated FN-C/H-V (8 µg/mL). Cell adhesion assays were terminated after 60 minutes, and cell adhesion was quantified as described above.
Endothelial Cell Spreading Assay
Glass coverslips (22x22 mm) were washed in 1:1 concentrated
sulfuric and nitric acid for 48 hours, rinsed extensively in deionized
water, and then dried. Coverslips were then placed at the bottom of
six-well tissue culture trays, and 2-mL aliquots of purified protein,
OVA-conjugated peptide, or OVA-conjugated OVA, diluted in PBS to a
concentration of 10 µg/mL, were added to each well. The
proteins/peptides were allowed to adsorb overnight at 37°C, after
which time nonspecific binding sites were blocked with 2 mg/mL OVA in
PBS. Cells were seeded onto the coated coverslip and allowed to
spread for 2 hours. Cells were fixed with 3.6% formaldehyde in PBS for
2 hours and then rinsed 3 times with PBS (5 minutes for each rinse).
Adherent cells were stained for 24 hours with 0.1% Coomassie brilliant
blue R-250 and then washed three times with a 20% methanol solution.
The cells were viewed and photographed on a Nikon Diaphot
phase-contrast microscope with a 40x objective, and the mean
spread-cell area was quantified by using OPTIMAS image
analysis software (BioScan Inc).
To assess the role of the sequence defined by FN-C/H-V in RHE-1A cell spreading on FN and the 33/66-kD fragments, RHE-1A cells were preincubated for 60 minutes at 37°C in 500, 100, 50, 10, and 5 µg/mL of FN-C/H-V or scrambled FN-C/H-V. These peptides were not conjugated to OVA. Cells treated in this manner were then added to Immulon I plates coated with 5 µg/mL FN, 5 µg/mL of the 33/66-kD fragments of FN, 8 µg/mL OVA-conjugated FN-C/H-V, 5 µg/mL type I collagen, or 5 µg/mL laminin. Spreading assays were terminated after 2 hours and stained as previously described. The cells were viewed and photographed on a Nikon Diaphot phase-contrast microscope with a 40x objective. Cells with an area >300 µm2 were considered to be spread. Spreading was expressed as the percentage of cells spread on the surface (counted in randomly chosen fields) for triplicate wells for each condition. Cell sample size was not <100.
Cell Migration Assay
Subconfluent endothelial cells were harvested by using
trypsin/EDTA and resuspended in basal medium containing 10% FBS. The
cells were counted, centrifuged, and resuspended twice in basal medium
containing 2 mg/mL OVA at a final density of 1x106
cells per milliliter. Intact FN, the 33/66-kD fragments of FN,
OVA-conjugated peptides, or OVA-conjugated OVA was diluted in basal
medium containing 2 mg/mL OVA, and 33-µL aliquots were added to the
lower chambers of a modified Boyden chamber (Neuroprobe). After
assembly of the Boyden chamber with a polycarbonate filter (pore size,
8 µm; Costar/Nucleopore) separating the upper and lower wells,
endothelial cells were added to the upper wells of the Boyden chamber
(5x104 cells per well). The chambers were then incubated
for 4 hours at 37°C in 5% CO2 in air, at which time the
filters were removed, fixed, and stained. Those cells that had not
migrated through the filter were removed with a cotton swab, and the
filters were mounted on glass slides for quantification using a Zeiss
photomicroscope equipped with an ocular grid. Migration was expressed
as the number of cells (counted in four randomly chosen fields, 25x
objective) that had migrated through the filter per unit area (square
millimeters) for triplicate wells for each condition.
Endothelial Cell Cytoskeletal Reorganization and Focal Contact
Formation
Cells were seeded onto glass coverslips coated with purified
proteins, OVA-conjugated peptides, or OVA-conjugated OVA, blocked as
previously described, and allowed to attach and spread for 3 hours, a
time point at which focal contact formation is believed to be
maximal.41 Cells were then fixed with 3.6%
formaldehyde/PBS solution for 15 minutes at room temperature. The cells
were permeabilized with 5% Triton X-100 in PBS for 5 minutes at room
temperature, washed three times with PBS, blotted dry, and covered with
a 25-µL aliquot of rhodamine phalloidin (Molecular Probes Inc). After
a 20-minute incubation at room temperature, the cells were washed twice
with PBS and mounted in a 1:1 solution of PBS/glycerol. The slides were
then viewed and photographed on a Nikon Diaphot phase-contrast
epifluorescence microscope with a 60x oil immersion objective.
To assess thaumatin (talin, Sigma) localization in focal contacts, cells were fixed and permeabilized in ice-cold methanol and ice-cold acetone, respectively, and then rinsed in dH2O. The cells were then covered with 10% normal goat serum (NGS) in PBS solution and incubated for 30 minutes at room temperature. The cells were then incubated with a 1:20 dilution of mouse monoclonal anti-talin (Sigma) in 10% NGS/PBS for 45 minutes at room temperature. The cells were next washed three times (5 minutes each) in 10% NGS/PBS. A 50 µg/mL anti-mouse IgG fluorescent conjugate (diluted in 10% NGS/PBS) was then added to each coverslip and incubated for 45 minutes at room temperature. The cells were then rinsed three times (5 minutes each) in PBS. Photographs were taken on a Nikon Diaphot epifluorescence microscope with a 60x objective. Focal contact formation was quantified by using OPTIMAS image analysis software (BioScan Inc).
To assess focal contact formation by interference reflectance microscopy (IRM), an acid-washed coverslip (24x30 mm) was sealed over a 20-mm-diameter opening in the bottom of a 50x10-mm polystyrene Petri dish (Falcon) by using a 1:1:1 paraffin/petroleum jelly/beeswax mixture. A 3-mL aliquot of purified protein or OVA-conjugated peptide, diluted in PBS to a concentration of 10 µg/mL, was added to the well. The proteins/peptides were allowed to adsorb overnight at 37°C, after which time nonspecific binding sites were blocked with 2 mg/mL OVA in PBS. Cells were then seeded onto coated coverslips and incubated as previously described.
IRM was performed with a Zeiss IM 35 inverted microscope (Carl Zeiss, Inc) with a 63x oil immersion Antiflex-NeoFluar objective, HBO 50-W mercury bulb, Zeiss H-D reflector, 2 FL housing, and a 546±2-nm filter. Photographs of IRM images of spread cells were taken 3 hours after seeding.
Statistical Analysis
All statistical analyses were carried out using
SIGMASTAT (Jandel Scientific Software).
One-way ANOVA in combination with the Bonferroni method of pairwise
comparisons was used to detect statistically significant differences in
RHE-1A cell adhesion and spreading on FN, the 33/66-kD fragments of FN,
or OVA-conjugated FN-C/H-V in the presence and absence of FN-C/H-V.
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Endothelial Cell Adhesion to FN, the 33/66-kD Fragments of FN, and Synthetic Peptides
RHE-1A cells adhered to surfaces coated with intact FN in a concentration-dependent manner, reaching maximal cell adhesion (77%) after 90 minutes at a coating concentration of 10 µg/mL (Fig 2
). RHE-1A cell adhesion to the 33/66-kD heparin-binding
fragments was also concentration dependent. Maximal cell adhesion
(80%) was observed at a coating concentration of 10 µg/mL (Fig 2).
To define in greater detail the adhesion-promoting domains within the
33/66-kD fragments, several chemically synthesized peptides
representing noncontiguous amino acid sequences from this
fragment with known adhesion-promoting activities were tested for their
ability to promote endothelial cell adhesion. The peptides FN-C/H-I,
FN-C/H-II, FN-C/H-III, FN-C/H-IV, and FN-C/H-V (Fig 1, Table 1) have
been shown previously to promote the adhesion of various cells in
culture.25 26 27 28 31 32 33 These peptides are cationic (net
charge, +2 to +4) and hydrophilic (hydropathy index, -23.7 to -29.3),
and they bind [3H]heparin. Scrambled FN-C/H-V,
which represents the randomized amino acid sequence of
FN-C/H-V, is also cationic and hydrophilic and has been included as a
control (Table 1). Peptide CS-1, present in the 33-kD but not the
66-kD fragment, also promotes cell adhesion and spreading of several
cell types.18 19 20 22 CS-1 is relatively hydrophobic
(hydropathy index, -9.9), and unlike the FN-C/H-peptides, it is
anionic (net charge, -4) and does not bind
[3H]heparin.25
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), 33/66-kD fragments (
), or ovalbumin (
). Cell
adhesion was measured as described in "Materials and Methods."
Data represent mean±1 SD (n=3).
RHE-1A cells adhered to surfaces coated with OVA-conjugated FN-C/H-I,
FN-C/H-II, FN-C/H-III, FN-C/H-IV, and FN-C/H-V in a
concentration-dependent manner (Fig 3). Maximal cell
adhesion to these peptides ranged from 65% (FN-C/H-V) to 80%
(FN-C/H-III) at coating concentrations of >5 µg/mL. RHE-1A cells
also adhered to surfaces coated with OVA-conjugated CS-1, although
adhesion was relatively poor (45% at a coating concentration of 100
µg/mL). Surfaces coated with OVA-conjugated OVA or OVA-conjugated
scrambled FN-C/H-V did not support endothelial cell adhesion,
regardless of the coating concentration tested.
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),
FN-C/H-V (
), CS-1 (
), scrambled FN-C/H-V (
), or
ovalbumin (
). Cell adhesion was measured as described in
"Materials and Methods." Data represent the mean of
triplicate determinants; the SD of each point was <10%.
Inhibition of RHE-1A Cell Adhesion to FN, the 33/66-kD Fragments of
FN, and OVA-Conjugated FN-C/H-V by FN-C/H-V
To assess the contribution of the sequence defined by FN-C/H-V to
the cell adhesion–promoting activity of FN and the 33/66-kD fragments
of FN, we next tested the ability of FN-C/H-V to inhibit RHE-1A cell
adhesion to surfaces coated with FN, the 33/66-kD fragments, or
OVA-conjugated FN-C/H-V (Fig 4). FN-C/H-V inhibited
RHE-1A cell adhesion to surfaces coated with OVA-conjugated FN-C/H-V in
a concentration-dependent manner. This inhibition was statistically
significant (P<.05) at all concentrations tested (Fig 4).
FN-C/H-V also inhibited cell adhesion to the 33/66-kD fragments of FN,
although this inhibition was statistically significant
(P<.05) only at the highest concentration tested (500
µg/mL). FN-C/H-V did not inhibit RHE-1A cell adhesion to surfaces
coated with FN. Scrambled FN-C/H-V did not inhibit RHE-1A cell adhesion
to surfaces coated with FN, the 33/66-kD fragments, or FN-C/H-V (Fig 4).
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Endothelial Cell Spreading on FN, the 33/66-kD Fragments of FN, and
Synthetic Peptides
RHE-1A cell adhesion to surfaces coated with intact FN or with the
33/66-kD fragments of FN was accompanied by cell spreading. Two hours
after seeding, the cells exhibited a well-spread morphology (Fig 5A and 5B). RHE-1A cell adhesion to surfaces coated with
OVA-conjugated FN-C/H-III was not accompanied by spreading (mean cell
area, 184.9±68.4 µm2) (Fig 5C), regardless of coating
concentration or incubation time. Cells also failed to spread on
OVA-conjugated FN-C/H-I, FN-C/H-II, FN-C/H-IV, CS-1, and scrambled
FN-C/H-V, as did cells on surfaces coated with OVA-conjugated OVA,
which was included as a negative control (data not shown). In contrast,
2 hours after seeding on surfaces coated with OVA-conjugated FN-C/H-V,
RHE-1A cells displayed a well-spread morphology (Fig 5D), similar to
that observed on intact FN and on the 33/66-kD fragments. Ninety-eight
percent of RHE-1A cells seeded on FN spread after 2 hours and had a
mean cell area of 1547±722 µm2. Ninety-four percent of
those cells seeded on the 33/66-kD fragments of FN were spread after 2
hours and had a mean cell area of 1015±557µm2. Finally,
85% of those cells seeded on FN-C/H-V were spread after 2 hours and
had a mean cell area of 1025±722 µm2.
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Inhibition of RHE-1A Cell Spreading on FN, the 33/66-kD Fragments
of FN, and OVA-Conjugated FN-C/H-V by FN-C/H-V
To assess the contribution of the sequence defined by FN-C/H-V to
the cell spreading/promoting activity of FN and the 33/66-kD fragments
of FN, we tested the ability of FN-C/H-V to inhibit RHE-1A cell
spreading on surfaces coated with FN, the 33/66-kD fragments, or
OVA-conjugated FN-C/H-V. FN-C/H-V (500 µg/mL) inhibited RHE-1A cell
spreading on surfaces coated with OVA-conjugated FN-C/H-V and on the
33/66-kD fragments of FN (Fig 6A). RHE-1A spreading on
surfaces coated with FN was also inhibited by FN-C/H-V (Fig 6A).
Scrambled FN-C/H-V (500 µg/mL) did not inhibit RHE-1A cell spreading
on FN, the 33/66-kD fragments, or OVA-conjugated FN-C/H-V (Fig 6A).
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To assess the specificity of the inhibitory effect of FN-C/H-V on
RHE-1A cell spreading, we then tested the ability of FN-C/H-V to
inhibit RHE-1A cell spreading on surfaces coated with the extracellular
matrix proteins, type I collagen, and laminin. FN-C/H-V did not inhibit
RHE-1A cell spreading on type I collagen or on laminin at any
concentration tested (Fig 6A). These results suggest that the
inhibitory effect of FN-C/H-V is not the result of a general inhibition
of RHE-1A cell spreading on any extracellular matrix protein but,
rather, is specific for FN and the FN-derived 33/66-kD fragments.
The inhibition of RHE-1A cell spreading on surfaces coated with
OVA-conjugated FN-C/H-V was concentration dependent (Fig 6B) and was
statistically significant (P<.05) at all concentrations
tested. FN-C/H-V also inhibited RHE-1A cell spreading on surfaces
coated with the 33/66-kD fragments of FN in a concentration-dependent
manner, and this inhibition was statistically significant
(P<.05) when FN-C/H-V was present at 100 and 500
µg/mL. RHE-1A cell spreading on surfaces coated with FN was also
inhibited by FN-C/H-V. This inhibition was concentration dependent and
statistically significant (P<.05) when FN-C/H-V was
present at 500 µg/mL. Scrambled FN-C/H-V did not inhibit RHE-1A
cell spreading on FN, the 33/66-kD fragments of FN, or OVA-conjugated
FN-C/H-V (data not shown).
Endothelial Cell Migration on FN, the 33/66-kD Fragments of FN, and
Synthetic Peptides
FN promoted the migration of RHE-1A cells in a
concentration-dependent manner, as did the 33/66-kD fragments of FN
(Fig 7). Migration was observed across polycarbonate
filters in response to FN and the 33/66-kD fragments at concentrations
of 
0.2 µmol/L and above. RHE-1A cells also migrated in response to
OVA-conjugated FN-C/H-V in a concentration-dependent manner (Fig 7), although this peptide was two orders of magnitude less potent than
either FN or the 33/66-kD fragments of FN. RHE-1A cell migration was
observed in the presence of OVA-conjugated FN-C/H-V at a concentration
of 10 µmol/L and above. RHE-1A cells did not migrate in response to
OVA-conjugated FN-C/H-III or OVA-conjugated scrambled FN-C/H-V (Fig 7)
or to OVA-conjugated FN-C/H-I, FN-C/H-II, FN-C/H-IV, CS-1, or OVA (data
not shown).
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Cytoskeletal Reorganization and Focal Contact Formation
Cell spreading is frequently accompanied by reorganization of the
cytoskeleton and the formation of actin stress fibers. RHE-1A cells
spread on intact FN and the 33/66-kD fragments exhibited a
well-organized actin cytoskeleton (Fig 8A and 8B).
Abundant actin stress fibers were seen traversing the cytoplasm,
terminating in the periphery of the cells. Cells spread on FN-C/H-V
also contained abundant actin stress fibers traversing the cytoplasm in
a pattern similar to that seen on FN and the 33/66-kD fragments (Fig 8C). In contrast, cells adherent to FN-C/H-III possessed a cortical
ring of actin but were largely devoid of actin stress fibers (Fig 8D).
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RHE-1A cells spread for 3 hours on FN displayed a uniform distribution
of large, randomly oriented focal contacts as assessed by IRM (Fig 9A). Focal contacts formed by RHE-1A cells on the
33/66-kD fragments were less numerous compared with those formed by
RHE-1A cells on FN but were similar in distribution (Fig 9B). In
contrast, focal contacts formed by RHE-1A cells on FN-C/H-V were much
less abundant than those formed by RHE-1A cells on FN or the 33/66-kD
fragments. Furthermore, focal contacts in RHE-1A cells on FN-C/H-V
appeared to be limited to the periphery of the cells (Fig 9C). Focal
contact formation was not observed in RHE-1A cells on FN-C/H-III (data
not shown).
|
Focal contacts contain the cytoskeletal protein talin.42 A
monoclonal antibody against talin was therefore used to confirm the
presence of focal contacts in RHE-1A cells spread on FN, the 33/66-kD
fragments, and FN-C/H-V. The distribution of talin-rich focal contacts
was similar to the pattern of focal contacts seen by IRM. The
percentage of cells displaying focal contacts was calculated by
previously described methods.41 RHE-1A cells spread on FN
displayed the highest percentage of focal contacts (93%) (Fig 10A). Focal contacts formed by RHE-1A cells spread on
the 33/66-kD fragments were reduced compared with those formed by
RHE-1A cells on FN, with 85% of the cells displaying focal contacts
(Fig 10B). Finally, 65% of RHE-1A cells spread on FN-C/H-V displayed
focal contacts (Fig 10C).
|
BAEC and HUVEC Adhesion, Spreading, and Migration on FN, the
33/66-kD Fragments of FN, and FN-C/H-V
Endothelial cells from different sites can exhibit different
functional properties. Therefore, we sought to determine whether the
adhesion-, spreading-, and motility-promoting activity of FN-C/H-V was
specific for the RHE-1A cell line. BAECs adhered to surfaces coated
with intact FN, the 33/66-kD fragments, or OVA-conjugated FN-C/H-V in a
concentration-dependent manner, reaching maximal levels of 81%, 83%,
and 40%, respectively, at a coating concentration of 10 µg/mL after
90 minutes (Fig 11). HUVECs also adhered to surfaces
coated with intact FN, the 33/66-kD fragments, or OVA-conjugated
FN-C/H-V in a concentration-dependent manner, reaching maximal levels
of 80%, 78%, and 63%, respectively, at a coating concentration of 2
µg/mL after 90 minutes (Fig 11). Furthermore, adhesion of both BAECs
and HUVECs to surfaces coated with intact FN, the 33/66-kD fragments,
or OVA-conjugated FN-C/H-V was accompanied by both cell spreading and
cell migration at a coating concentration of 100 µg/mL (Table 2).
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|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FN plays an important role in endothelial cell adhesion, spreading, and motility.6 7 A number of functionally distinct domains of FN have been identified (Fig 1
), including several
domains that support cell adhesion.5 The RGDS tetrapeptide
sequence is the most widely known.9 Vascular endothelial
cells adhere to a fragment of FN containing this
sequence,10 and Hayman et al11 have shown
that an RGDS-containing synthetic peptide can inhibit vascular
endothelial cell adhesion to intact FN. However, the 33- and 66-kD
heparin-binding fragments from the carboxyl-terminal third of the A
chain and B chain of the FN molecule, respectively, also support the
adhesion of various cell types,24 including
HUVECs.10 In the present study, we demonstrate that the 33/66-kD fragments of FN promote the adhesion of vascular endothelial cells from several sources. In addition, we demonstrate that vascular endothelial cell adhesion to the 33/66-kD fragments of FN is accompanied by a cascade of cellular events, including cell spreading, cytoskeletal reorganization, focal contact formation, and ultimately cell migration. These processes are critical to the maintenance of vascular integrity and to vessel repair after injury43 44 and were previously associated only with the RGDS-containing central cell binding domain of FN.3 These findings are consistent with previous reports demonstrating that the 33/66-kD fragments promote cell adhesion independent of the central cell binding domain.10 24 Finally, we have taken the first step toward defining the molecular basis of endothelial cell adhesion-, spreading-, and motility-promoting activity of the 33/66-kD fragments by identifying several amino acid sequences within these fragments that promote endothelial cell adhesion. One of these peptides (FN-C/H-V) also supports endothelial cell spreading and motility.
RHE-1A cells adhered to each of the peptides tested in the present study. These cells differ from other cell types that recognize specific subsets of these peptide sequences. Rabbit corneal epithelial cells, for example, adhered to FN-C/H-I, FN-C/H-III, FN-C/H-IV, and FN-C/H-V but did not adhere to FN-C/H-II or CS-1.31 32 Human keratinocytes, in contrast, adhered to FN-C/H-II, FN-C/H-V, and CS-1 but not to FN-C/H-III or FN-C/H-IV.27 33 These findings reinforce the view that cell adhesion to the 33/66-kD fragments involves multiple adhesion-promoting domains and that the adhesion of a specific cell type to the 33/66-kD fragments is the result of interactions with a number of peptide domains. Our results also demonstrate that FN-C/H-V possesses unique activities not common to all peptides from these domains of FN, since only peptide FN-C/H-V promoted endothelial cell spreading and migration. This is consistent with reports demonstrating that only FN-C/H-V promoted the spreading and migration of rabbit corneal epithelial cells32 and the spreading of human keratinocytes.33
RHE-1A cells also adhere to CS-1, a peptide whose sequence is found in
the alternatively spliced (type IIIcs) region of the A chain of FN.
Conforti et al21 and Wayner et al22 have
shown that T-lymphocyte adhesion to CS-1 is mediated by the
4ß1 integrin but that this integrin is not
expressed by HUVECs. However, Massia and Hubbell23 have
recently reported that HUVECs adhere to CS-5, a second cell adhesion
site in the IIIcs region of FN, and to shorter peptides containing the
REDV sequence, which is the minimal recognition signal in CS-5, via the
4ß1 integrin. We have not examined
4ß1 integrin expression in RHE-1A cells
and therefore do not know if RHE-1A cell adhesion to CS-1 is mediated
by this receptor or by an as-yet-unidentified receptor.
Peptide FN-C/H-V contributed to the adhesion-, spreading-, and migration-promoting activity of the 33/66-kD fragments of FN. This conclusion is based on several lines of evidence. First, we have demonstrated that FN-C/H-V directly promotes the adhesion, spreading, and migration of RHE-1A, BAECs, and HUVECs and therefore mimics the 33/66-kD fragments of FN in this regard. Second, FN-C/H-V (but not scrambled FN-C/H-V) inhibited RHE-1A cell spreading in a concentration-dependent manner on FN and the 33/66-kD fragments and, to a lesser extent, the adhesion of RHE-1A cells to the 33/66-kD fragments of FN. The failure of soluble FN-C/H-V to completely block RHE-1A cell adhesion to the 33/66-kD fragments of FN is not surprising, since at least five distinct peptide sequences participate in endothelial cell adhesion to the 33/66-kD fragments, consistent with previous reports describing multiple active domains within the 33/66-kD fragments.17 25 26 27 28 36 Soluble FN-C/H-V did not effectively block RHE-1A adhesion to FN. This is also not surprising, since not only are additional cell adhesion–promoting sites available within the 33/66-kD fragments but the RGDS adhesion–promoting sequence in FN is also present.9 The ability of FN-C/H-V to inhibit RHE-1A spreading on the 33/66-kD fragments is consistent with the fact that FN-C/H-V was the only sequence to promote RHE-1A spreading.
Cell spreading is frequently accompanied by cytoskeletal reorganization, and indeed, cells spread on FN-C/H-V exhibited an actin cytoskeletal network that was similar to that formed by cells on the 33/66-kD fragments or on FN. Furthermore, RHE-1A cell spreading on peptide FN-C/H-V did not require de novo protein synthesis, since RHE-1A cells treated with cycloheximide displayed spreading on the 33/66-kD fragments and FN-C/H-V and an actin cytoskeleton similar to that seen in the absence of cycloheximide (data not shown). We have also shown that the 33/66-kD fragments of FN and FN-C/H-V promoted the adhesion, spreading, and migration of endothelial cells from bovine aorta and human umbilical vein. These findings suggest that the ability of the 33/66-kD fragments and FN-C/H-V to support cell adhesion and spreading is shared by endothelial cells from diverse species and anatomic locations.
RHE-1A cells spread on FN-C/H-V also form focal contacts. However,
focal contact formation was less extensive than on FN or 33/66-kD
fragments. In addition, focal contacts were limited to the periphery of
the cells. These differences may be due to the fact that the 33/66-kD
fragments contain the additional sequences of FN-C/H-I, FN-C/H-II,
FN-C/H-III, FN-C/H-IV, and CS-1 compared with intact FN, which contains
the 33/66-kD fragments plus the RGD-containing central cell-binding
domain and possibly other sites as well. Cell interaction with multiple
sites may affect the localization of specific adhesion receptors
(integrins and proteoglycans) and associated cytoplasm proteins (such
as talin, vinculin, and -actinin) at the points of cytoskeleton
organization. These events may ultimately be reflected in differences
in the number and distribution of focal contacts.
The cell adhesion receptor(s) for FN-C/H-V has not been identified. The ability of this peptide to bind heparin, a trait that is shared by the 33/66-kD fragments of FN, suggests a role for proteoglycans in cell adhesion to this peptide. Proteoglycans have been implicated in cell adhesion to several peptides from the 33/66-kD fragments,29 45 and Woods et al46 have recently suggested that in human fibroblasts the receptor for FN-C/H-V is indeed a heparan sulfate proteoglycan. It is unclear whether endothelial cells also interact with FN-C/H-V via a heparan sulfate proteoglycan or perhaps with other adhesion receptors such as integrins. Although the identity of this receptor(s) remains unknown, its interactions with FN-C/H-V clearly involve signaling events that culminate in the reorganization of the actin cytoskeleton, the formation of focal contacts, and cell migration. Identification of the FN-C/H-V receptor(s) will be crucial to understanding the mechanism of action of this peptide.
In conclusion, these studies demonstrate that the 33/66-kD fragments and peptides from these fragments can support endothelial cell adhesion. One peptide (FN-C/H-V) also supported cytoskeletal reorganization, spreading, and migration of vascular endothelial cells from various sources. Furthermore, this peptide has been shown to have effects on endothelial cell cytoskeletal organization that mimic those seen in cells on the 33/66-kD fragments and FN. FN-C/H-V inhibited RHE-1A cell spreading on FN and the 33/66-kD fragments in a concentration-dependent manner. FN-C/H-V had a modest effect on RHE-1A cell adhesion to the 33/66-kD fragments of FN and no effect on RHE-1A cell adhesion to FN. These findings provide evidence that peptide FN-C/H-V is unique among this group of peptides derived from the 33/66-kD heparin-binding fragments of FN in its ability to promote the adhesion, spreading, and motility of vascular endothelial cells and suggest an important role for FN-C/H-V in endothelial cell interactions with the 33/66-kD fragments of FN.
|
Acknowledgments |
|---|
This study was supported by the University of Minnesota Center for Interfacial Engineering (Dr Mooradian) and a grant from the Minnesota Medical Foundation (Dr Mooradian). This manuscript partially fulfills the requirements of the University of Minnesota Graduate School for a master's thesis (J.C. Huebsch).
Received September 1, 1994; accepted March 20, 1995.
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