Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 1995;77:43-53

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huebsch, J. C.
Right arrow Articles by Mooradian, D. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huebsch, J. C.
Right arrow Articles by Mooradian, D. L.
(Circulation Research. 1995;77:43-53.)
© 1995 American Heart Association, Inc.

Articles

Endothelial Cell Interactions With Synthetic Peptides From the Carboxyl-Terminal Heparin-Binding Domains of Fibronectin

Joseph C. Huebsch, James B. McCarthy, Clement A. Diglio, Daniel L. Mooradian

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.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 1Down, Table 1Down), 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 {alpha}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



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. Diagram showing location of synthetic peptides within fibronectin (FN). The proteolytic cleavage of plasma fibronectin with trypsin (T) and cathepsin D (C) yields a number of domains including the following: a weak heparin binding domain (I), a collagen-binding (noncovalent) domain (II), the RGDS-containing central cell-binding domain (IV), the carboxyl-terminal heparin-binding and cell adhesion–promoting domain (V), and free sulfhydryl–containing domains (III and VI). The approximate locations of the peptides FN-C/H-I, FN-C/H-II, FN-C/H-III, FN-C/H-IV, FN-C/H-V, and CS-1 (A chain only) within the 33/66-kD fragment are also shown.


View this table:
[in this window]
[in a new window]
  		Table 1. Synthetic Peptides From the 33/66-kD Fragments of Fibronectin

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 {alpha}4ß1 integrin.20 21 Several studies indicate that human umbilical vein endothelial cells (HUVECs) do not express the {alpha}4ß1 integrin,21 22 but a recent study by Massia and Hubbell23 suggests that HUVECs do express the {alpha}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.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 {cong}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 1Up and Table 1Up. 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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 2Down). 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 2Down). 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 1Up, Table 1Up) 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 1Up). 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



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. Graph showing adhesion of RHE-1A cells to surfaces coated with fibronectin and the 33/66-kD fragments. Radiolabeled RHE-1A cells were incubated for 1 hour on surfaces coated with fibronectin ({circ}), 33/66-kD fragments ({bullet}), or ovalbumin ({square}). 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 3Down). 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.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 3. Graph showing adhesion of RHE-1A cells to surfaces coated with FN-C/H peptides. Radiolabeled RHE-1A cells were incubated for 1 hour on surfaces coated with ovalbumin conjugates of FN-C/H-I ({circ}), FN-C/H-II ({bullet}), FN-C/H-III ({square}), FN-C/H-IV ({blacksquare}), FN-C/H-V ({triangleup}), CS-1 ({blacktriangleup}), scrambled FN-C/H-V ({blacktriangledown}), or ovalbumin ({triangledown}). 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 4Down). 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 4Down). 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 4Down).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Graph showing inhibition of RHE-1A cell adhesion by FN-C/H-V. Immulon-I plates (Dynatech Laboratories, Inc) were coated with fibronectin (5 µg/mL; {circ}, {bullet}), the 33/66-kD fragments of fibronectin (5 µg/mL; {square}, {blacksquare}), or ovalbumin-conjugated FN-C/H-V (8 µg/mL; {triangleup}, {blacktriangleup}). RHE-1A cells were preincubated for 60 minutes with either 500, 100, 50, 10, or 5 µg/mL of FN-C/H-V ({circ}, {square}, {triangleup}) or scrambled FN-C/H-V ({bullet}, {blacksquare}, {blacktriangleup}) and then added to the plates. Data represent the mean of triplicate determinants; the SD of each point was <10%. *Statistically significant difference between control and treated groups (P<.05) as determined by one-way ANOVA followed by pairwise multiple comparisons using the Bonferroni t test.

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 5ADown and 5BDown). 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 5CDown), 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 5DDown), 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.



View larger version (83K):
[in this window]
[in a new window]
  		Figure 5. Photomicrographs showing spreading of RHE-1A cells on surfaces coated with fibronectin (A), the 33/66-kD fragments (B), ovalbumin-conjugated FN-C/H-III (C), and ovalbumin-conjugated FN-C/H-V (D). Bar=10 µm.

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 6ADown). RHE-1A spreading on surfaces coated with FN was also inhibited by FN-C/H-V (Fig 6ADown). 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 6ADown).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. A, Bar graph showing RHE-1A cells preincubated for 60 minutes with 500 µg/mL FN-C/H-V (solid bars) or scrambled FN-C/H-V (hatched bars) before seeding onto Immulon-I plates (Dynatech Laboratories, Inc) coated with fibronectin (FN, 5 µg/mL), the 33/66-kD fragments of FN (5 µg/mL), ovalbumin-conjugated FN-C/H-V (8 µg/mL), type I collagen (5 µg/mL), or laminin (5 µg/mL). Control RHE-1A cells were not treated with peptides before seeding (open bars). Adhesion assays were terminated after 60 minutes, and cell spreading was quantified as described in "Materials and Methods." B, Graph showing RHE-1A cells preincubated for 60 minutes with 5, 10, 50, 100, or 500 µg/mL FN-C/H-V ({circ}, {square}, {triangleup}) or scrambled FN-C/H-V ({bullet}, {blacksquare}, {blacktriangleup}) before seeding on Immulon-I plates coated with FN ({circ}, {bullet}), the 33/66-kD fragments of FN ({square}, {blacksquare}),or ovalbumin-conjugated FN-C/H-V ({triangleup}, {blacktriangleup}). Cells were counted in four randomly chosen fields in each of three wells for each condition. The sample size (ie, number of cells analyzed) was not <100 for each condition. *Statistically significant difference between control and treated groups (P<.05) as determined by one-way ANOVA followed by multiple pairwise comparisons using the Bonferroni method.

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 6AUp). 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 6BUp) 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 7Down). Migration was observed across polycarbonate filters in response to FN and the 33/66-kD fragments at concentrations of {approx}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 7Down), 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 7Down) or to OVA-conjugated FN-C/H-I, FN-C/H-II, FN-C/H-IV, CS-1, or OVA (data not shown).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 7. Graph showing migration of RHE-1A cells in response to fibronectin ({circ}), 33/66-kD fragments ({bullet}), ovalbumin-conjugated FN-C/H-V ({square}), or ovalbumin-conjugated scrambled FN-C/H-V ({blacksquare}). Cells were allowed to migrate for 4 hours and were then fixed, stained, and quantified as described in "Materials and Methods." Data represent mean±1 SD (n=3).

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 8ADown and 8BDown). 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 8CDown). In contrast, cells adherent to FN-C/H-III possessed a cortical ring of actin but were largely devoid of actin stress fibers (Fig 8DDown).



View larger version (53K):
[in this window]
[in a new window]
  		Figure 8. Actin cytoskeletal organization by RHE-1A cells spread on fibronectin (A), the 33/66-kD fragments (B), ovalbumin-conjugated FN-C/H-III (C), and ovalbumin-conjugated FN-C/H-V (D) visualized by using a rhodamine phalloidin conjugate as described in "Materials and Methods." Bar=10 µm.

RHE-1A cells spread for 3 hours on FN displayed a uniform distribution of large, randomly oriented focal contacts as assessed by IRM (Fig 9ADown). 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 9BDown). 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 9CDown). Focal contact formation was not observed in RHE-1A cells on FN-C/H-III (data not shown).



View larger version (64K):
[in this window]
[in a new window]
  		Figure 9. Focal contact formation by RHE-1A cells spread on fibronectin (A), 33/66-kD fragments (B), and ovalbumin-conjugated FN-C/H-V (C). Visualization was by interference reflectance microscopy as described in "Materials and Methods." Bar=10 µm.

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 10ADown). 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 10BDown). Finally, 65% of RHE-1A cells spread on FN-C/H-V displayed focal contacts (Fig 10CDown).



View larger version (45K):
[in this window]
[in a new window]
  		Figure 10. Focal contact formation by RHE-1A cells spread on fibronectin (A), 33/66-kD fragments (B), and ovalbumin-conjugated FN-C/H-V (C). Visualization was by thaumatin (talin) localization as described in "Materials and Methods." Bar=10 µm.

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 11Down). 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 11Down). 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 2Down).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 11. Graphs showing adhesion of bovine aortic endothelial cells (A) and human umbilical vein endothelial cells (B) to surfaces coated with fibronectin ({blacktriangleup}), the 33/66-kD fragments ({circ}), ovalbumin-conjugated FN-C/H-III ({bullet}), ovalbumin-conjugated FN-C/H-V ({square}), or ovalbumin-conjugated ovalbumin ({blacksquare}). Cell adhesion was measured as described in "Materials and Methods." Data represent the mean of triplicate determinants; the SD of each point was <10%.


View this table:
[in this window]
[in a new window]
  		Table 2. Bovine Aortic Endothelial Cell and Human Umbilical Vein Endothelial Cell Adhesion, Spreading, and Migration on Fibronectin, the 33/66-kD fragments of Fibronectin, and FN-C/H-V


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
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 1Up), 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 {alpha}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 {alpha}4ß1 integrin. We have not examined {alpha}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 {alpha}-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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Furcht LT. Critical factors controlling angiogenesis: cell products, cell matrix, and growth factors. Lab Invest. 1986;55:505-509. [Medline] [Order article via Infotrieve]
  2. Madri JA, Pratt BM, Tucker AM. Phenotypic modulation of endothelial cells by transforming growth factor-ß1 depends upon the composition and organization of the extracellular matrix. J Cell Biol. 1988;106:1375-1384. [Abstract/Free Full Text]
  3. Dejana E, Colella S, Conforti G, Abbadini M, Gaboli M, Marchisio PC. Fibronectin and vitronectin regulate the organization of their respective Arg-Gly-Asp adhesion receptors in cultured human endothelial cells. J Cell Biol. 1988;107:1215-1223. [Abstract/Free Full Text]
  4. Kramer RH, Fuh GM, Karasek MA. Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane. Biochemistry. 1985;24:7423-7430. [Medline] [Order article via Infotrieve]
  5. Hynes RO. Fibronectins. New York, NY: Springer-Verlag; 1990.
  6. Macara EJ, Howard PS. Adhesion of endothelial cells to extracellular matrix proteins. J Cell Physiol. 1983;116:76-86. [Medline] [Order article via Infotrieve]
  7. Madri J, Pratt BM, Yannariello-Brown J. Matrix-driven cell size change modulates aortic endothelial cell proliferation and sheet migration. Am J Pathol. 1988;132:18-27. [Abstract]
  8. Sekiguchi K, Fukuda M, Hakomori S-J. Domain structure of hamster plasma fibronectin. J Biol Chem. 1981;256:6452-6462. [Free Full Text]
  9. Pierschbacher MD, Ruoslahti E. The cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 1984;309:30-33. [Medline] [Order article via Infotrieve]
  10. Visser MR, Vercellotti GM, McCarthy JB, Goodman JL, Herbst TJ, Furcht LT, Jacob HS. Herpes simplex virus inhibits endothelial cell attachment and migration to extracellular matrix proteins. Am J Pathol. 1989;134:223-230. [Abstract]
  11. Hayman EG, Pierschbacher MD, Ruoslahti E. Detachment of cells from culture substrate by soluble fibronectin peptides. J Cell Biol. 1985;100:1948-1954. [Abstract/Free Full Text]
  12. Pytela R, Pierschbacher MD, Ruoslahti E. Identification and isolation of a 140kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell. 1985;40:191-198. [Medline] [Order article via Infotrieve]
  13. Obara M, Kang MS, Yamada KM. Site-directed mutagenesis of the cell binding domain of human fibronectin: separable, synergistic sites mediated adhesive function. Cell. 1988;53:649-657. [Medline] [Order article via Infotrieve]
  14. Aota S, Nagai T, Yamada KM. Characterization of regions of fibronectin besides the arginine-glycine-aspartic acid sequence required for adhesive function of the cell binding domain using site directed mutagenesis. J Biol Chem. 1991;266:15938-15943. [Abstract/Free Full Text]
  15. Nagai T, Yamakawa N, Aota S, Yamada SS, Olden K, Yamada KM. Monoclonal antibody characterization of two distinct sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration and matrix assembly. J Cell Biol. 1991;114:1295-1305. [Abstract/Free Full Text]
  16. Kornblihtt AR, Umezawa K, Vibe-Pedersen K, Baralle FE. Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J. 1985;4:1755-1759. [Medline] [Order article via Infotrieve]
  17. Humphries MJ, Akiyama SK, Komoriya A, Olden K, Yamada KM. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. J Cell Biol. 1986;103:2637-2647. [Abstract/Free Full Text]
  18. Humphries MJ, Akiyama SK, Komoriya A, Olden K, Yamada KM. Identification of two distinct regions of the type IIIcs connecting segment of human plasma fibronectin that promote cell type-specific adhesion. J Biol Chem. 1987;262:6886-6892. [Abstract/Free Full Text]
  19. Komoriya A, Green LJ, Mervic M, Yamada SS, Yamada KM, Humphries MJ. The minimal essential sequence for a major cell type specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid valine. J Biol Chem. 1991;266:15075-15079.[Abstract/Free Full Text]
  20. Mould AP, Komoriya A, Wayner EA, Yamada KM, Humphries MJ. The CS5 peptide is a second site in IIICS region of fibronectin recognized by the integrin {alpha}4ß1: Inhibition of {alpha}4ß1 function by RGD peptide homologues. J Biol Chem. 1991;266:3579-3585. [Abstract/Free Full Text]
  21. Conforti G, Dominguez-Jimenez C, Zanetti A, Gimbrone MA, Cremona O, Marchisio PC, Dejana E. Human endothelial cells express integrin receptors on the luminal aspect of their membrane. Blood. 1992;80:437-446. [Abstract/Free Full Text]
  22. Wayner EA, Garcia-Pardo A, Humphries MJ, McDonald JA, Carter WG. Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin. J Cell Biol. 1989;109:1321-1330. [Abstract/Free Full Text]
  23. Massia SP, Hubbell JA. Vascular endothelial cell adhesion and spreading promoted by the peptide REDV of the IIICS region of plasma fibronectin is mediated by integrin {alpha}4ß1. J Biol Chem. 1992;267:14019-14026. [Abstract/Free Full Text]
  24. McCarthy JB, Hagen ST, Furcht LT. Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells. J Cell Biol. 1986;102:179-188. [Abstract/Free Full Text]
  25. McCarthy JB, Skubitz APN, Zhao Q, Yi X-Y, Mickelson DJ, Klein DJ, Furcht LT. RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin- dependent and -independent activities. J Cell Biol. 1990;110:777-787. [Abstract/Free Full Text]
  26. Haugen PK, McCarthy JB, Skubitz APN, Furcht LT, Letourneau PC. Recognition of the A chain carboxy-terminal heparin binding region of fibronectin involves multiple sites: two contiguous sequences act independently to promote neural cell adhesion. J Cell Biol. 1990;111:2733-2745.[Abstract/Free Full Text]
  27. Wilke MS, Skubitz APN, Furcht LT, McCarthy JB. Human keratinocytes adhere to two distinct heparin-binding synthetic peptides derived from fibronectin. J Invest Dermatol. 1991;97:573-579. [Medline] [Order article via Infotrieve]
  28. McCarthy JB, Chelberg MK, Mickelson DJ, Furcht LT. Localization and chemical synthesis of fibronectin peptides with melanoma adhesion and heparin binding activities. Biochemistry. 1988;27:1380-1388. [Medline] [Order article via Infotrieve]
  29. Drake SL, Klein DJ, Mickelson DJ, Oegema TR, Furcht LT, McCarthy JB. Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide. J Cell Biol. 1992;117:1331-1341. [Abstract/Free Full Text]
  30. Iida J, Skubitz APN, Furcht LT, Wayner EA, McCarthy JB. Coordinate role for cell surface chondroitin sulfate proteoglycan and {alpha}4ß1 integrin in mediating melanoma cell adhesion to fibronectin. J Cell Biol. 1992;118:431-444. [Abstract/Free Full Text]
  31. Mooradian DL, McCarthy JB, Cameron DJ, Skubitz APN, Furcht LT. Rabbit corneal epithelial cells adhere to two distinct heparin binding synthetic peptides derived from fibronectin. Invest Ophthalmol Vis Sci. 1992;33:3034-3040. [Abstract/Free Full Text]
  32. Mooradian DL, McCarthy JB, Skubitz APN, Cameron DJ, Furcht LT. Characterization of FN-C/H-V, a novel synthetic peptide from fibronectin that promotes rabbit corneal epithelial cell adhesion, spreading, and motility. Invest Ophthalmol Vis Sci. 1993;34:153-164. [Abstract/Free Full Text]
  33. Wilke MS, Vespa J, Skubitz APN, Furcht LT, McCarthy JB. Human keratinocytes adhere and spread on synthetic peptide FN-C/H-V derived from fibronectin. J Invest Dermatol. 1993;101:43-48. [Medline] [Order article via Infotrieve]
  34. Diglio CA, Grammas P, Giacomelli F, Wiener J. Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization. Tissue Cell. 1988;20:477-492. [Medline] [Order article via Infotrieve]
  35. Gospodarowicz D, Moran J, Braun D, Birdwell C. Clonal growth of bovine vascular endothelial cells: fibroblast growth factor as a survival agent. Proc Natl Acad Sci U S A. 1976;73:4120-4124. [Abstract/Free Full Text]
  36. Mooradian DL, Weatherbee J, Lucas R, Furcht LT. Transforming growth factor-ß1 binds to immobilized fibronectin. J Cell Biochem. 1990;41:189-200.
  37. Laemmli UK. Cleavage and structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685. [Medline] [Order article via Infotrieve]
  38. Koliakos GG, Kouzi-Koliakos K, Furcht LT, Reger LA, Tsilibary EC. The binding of heparin to type IV collagen: domain specificity with identification of peptide sequences from the {alpha}1(IV) and {alpha}2(IV) chain which preferentially bind heparin. J Biol Chem. 1989;264:2313-2323. [Abstract/Free Full Text]
  39. Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982;157:105-132. [Medline] [Order article via Infotrieve]
  40. Danilov YN, Juliano RL. (Arg-Gly-Asp)n-albumin conjugates as a model substratum for integrin-mediated cell adhesion. Exp Cell Res. 1989;182:186-196. [Medline] [Order article via Infotrieve]
  41. Woods A, Couchman JR. Protein kinase C involvement in focal adhesion formation. J Cell Sci. 1992;101:1-14. [Free Full Text]
  42. Burridge K, Connell L. A new protein of adhesion plaques and ruffling membranes. J Cell Biol. 1983;97:359-367. [Abstract/Free Full Text]
  43. Wong MKK, Gotlieb AI. In vitro reendothelialization of a single-cell wound. Lab Invest. 1984;51:75-81. [Medline] [Order article via Infotrieve]
  44. Haudenschild CC, Schwartz SM. Endothelial regeneration: restitution of endothelial continuity. Lab Invest. 1979;41:407-418. [Medline] [Order article via Infotrieve]
  45. Haugen PK, Letourneau PC, Drake SL, Furcht LT, McCarthy JB. A cell-surface heparan sulfate proteoglycan mediates neural cell adhesion and spreading on a defined sequence from the c-terminal cell and heparin binding domain of fibronectin, FN-C/H-II. J Neurosci. 1992;12:2597-2608. [Abstract]
  46. Woods A, McCarthy JB, Furcht LT, Couchman JR. Selective ability of synthetic peptides from the COOH-terminal heparin-binding domain of fibronectin to promote focal adhesion formation. Mol Biol Cell. 1993;4:605-613.[Abstract]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
R. Gendelman, N. I. Burton-Wurster, J. N. MacLeod, and G. Lust
The Cartilage-specific Fibronectin Isoform Has a High Affinity Binding Site for the Small Proteoglycan Decorin
J. Biol. Chem., March 21, 2003; 278(13): 11175 - 11181.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page