Identification of the {alpha}v{beta}3 Integrin-interacting Motif of {beta}ig-h3 and Its Anti-angiogenic Effect*

Ju-Ock Nam {ddagger}, Jung-Eun Kim {ddagger}, Ha-Won Jeong {ddagger}, Sung-Jin Lee {ddagger}, Byung-Heon Lee {ddagger}, Je-Yong Choi {ddagger}, Rang-Woon Park {ddagger}, Jae Yong Park {ddagger} § and In-San Kim {ddagger} 

From the {ddagger}Cell and Matrix Biology National Research Laboratory, Department of Biochemistry, and the §Department of Internal Medicine, Kyungpook National University School of Medicine, Taegu 700-422, Korea

Received for publication, January 13, 2003 , and in revised form, April 11, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{beta}ig-h3 is an extracellular matrix protein that mediates adhesion and migration of several cell types through interaction with integrins. In the present study, we tested whether {beta}ig-h3 mediates endothelial cell adhesion and migration, thereby regulating angiogenesis. In this study, we demonstrate that not only {beta}ig-h3 itself but also all four fas-1 domains of {beta}ig-h3 mediate endothelial cell adhesion and migration through interaction with the {alpha}v{beta}3 integrin. We found that the {alpha}v{beta}3 integrin-interacting motif of the four fas-1 domains of {beta}ig-h3 is the same YH motif that we reported previously to interact with {alpha}v{beta}5 integrin. The YH peptide inhibited endothelial cell adhesion and migration in a dose-dependent manner. We demonstrate that the YH peptide has anti-angiogenic activity in vitro and in vivo using an endothelial cell tube formation assay and a Matrigel plug assay, respectively. Our results reveal that {beta}ig-h3 bears {alpha}v{beta}3 integrin-interacting motifs that mediate endothelial cell adhesion and migration and, therefore, may regulate angiogenesis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The {beta}ig-h3 is an extracellular matrix protein whose expression is induced by transforming growth factor-{beta} in several cell types (1). The {beta}ig-h3 protein is composed of 683 amino acids containing four homologous internal repeat domains. These domains are homologous to similar motifs in the Drosophila protein fasciclin-I and thus are denoted fas-1 domains. The fas-1 domain has highly conserved sequences found in the secretory and membrane proteins of many organisms, including mammals, insects, sea urchins, plants, yeast, and bacteria. The fas-1 domain interacts with other matrix proteins such as fibronectin, collagen, and laminin (2) and mediates cell adhesion and migration through interaction with integrins (3, 4). We have reported previously that it bears motifs interacting with the integrins {alpha}3{beta}1 (3) and {alpha}v{beta}5 (4). The fas-1 domain is also known to be involved in cell growth, differentiation, tumorigenesis, wound healing, and apoptosis (58).

Although {beta}ig-h3 mediates the adhesion of many different cell types, including corneal epithelial cells, chondrocytes, and fibroblasts, it is not known whether {beta}ig-h3 mediates endothelial cell adhesion and migration, thereby regulating angiogenesis. In this study, we demonstrate that {beta}ig-h3 mediates endothelial cell adhesion and migration through {alpha}v{beta}3 integrin and that the responsible motif is the YH motif, which has been reported to interact with the {alpha}v{beta}5 integrin. In addition, we show that the YH peptide inhibits endothelial tube formation and reduces the number of blood vessels in a Matrigel plug assay. Collectively, the YH motif of the fas-1 domains of the {beta}ig-h3 protein interacts with {alpha}v{beta}3 integrin and is an effective inhibitor of angiogenesis. Our data suggest that the peptide fragment containing the YH motif could be a drug candidate for the treatment of diseases dependent on angiogenesis.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA Constructs and Synthetic Peptides—Bacterial expression vectors for wild-type {beta}ig-h3, each fas-1 domain, and relevant mutants have been described previously (4). Recombinant {beta}ig-h3 proteins were induced and purified as described previously (4).

Cell Culture—Human umbilical vein endothelial cells (HUVECs)1 were cultured at 37 °C in 5% CO2 in EGM medium (Clontech) supplemented with 2% fetal bovine serum. HEK293 cells stably transfected with an empty vector (pcDNA3) or a human integrin {beta}3 expression vector were kindly provided by Dr. Jeffrey Smith (Burnham Institute, San Diego). These stable cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics.

Cell Adhesion Studies—The cell adhesion assay was performed as described previously (4). Briefly, flat-bottomed 96-well enzyme-linked immunosorbent assay plates (Costar, Corning Inc., NY) were incubated overnight at 4 °C with 10 µg/ml of recombinant {beta}ig-h3 proteins and then blocked for 1 h at room temperature with phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA). Cells were suspended in medium at a density of 3 x 105 cells/ml, and 0.1 ml of the cell suspension was added to each well of the coated plates. After incubation for 30 min at 37 °C, unattached cells were removed by rinsing once with PBS. Attached cells were then incubated for 1 h at 37 °C in 50 mM citrate buffer, pH 5.0, containing 3.75 mM p-nitrophenyl-N-acetyl-D-glycosaminide and 0.25% Triton X-100. Enzyme activity was blocked by adding 50 mM glycine buffer, pH 10.4, containing 5 mM EDTA, and the absorbance was measured at 405 nm in a Bio-Rad model 550 microplate reader.

Inhibition Assay—Synthetic peptides purchased from AnyGen Co. Ltd. (Kwangju, Korea) were tested for their ability to inhibit cells from adhering to the protein substrates coating the wells. The cell adhesion assay was performed in the presence or absence of the indicated concentrations of each peptide. To identify the receptor for {beta}ig-h3, monoclonal antibodies specific to different types of integrins (Chemicon, Temecula, CA) were preincubated (5 µg/ml) at 37 °C for 30 min with HUVEC in 0.1 ml of the cell suspension (3 x 105 cells/ml). The cells were then transferred onto plates precoated with recombinant {beta}ig-h3 proteins and incubated for 30 min at 37 °C. The attached cells were then quantified as described above. Function-blocking monoclonal antibodies to the following integrin subunits were used: {alpha}3 (P1B5), {alpha}5 (P1D6), {alpha}v (P3G8), {beta}1 (6S6), {beta}3 (B3A), {alpha}v{beta}3 (LM609), and {alpha}v{beta}5 (P1F6).

Migration Assay—Cell migration assays were performed in transwell plates (8 µm pore size, Costar, Cambridge, MA). The undersurface of the membrane was coated with 10 µg/ml recombinant {beta}ig-h3 proteins at 4 °C and then blocked for 1 h at room temperature with phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA). Cells were suspended in medium at a density of 3 x 105 cells/ml, and 0.1 ml of the cell suspension was added to the upper compartment of the filter with or without the indicated concentrations of each peptide. In some experiments, cells were preincubated at 37 °C for 30 min with anti-{alpha}v{beta}3 (LM609) or {alpha}v{beta}5 (P1F6). Cells were allowed to migrate for 6–8 h at 37 °C. Migration was terminated by removing the cells from the upper compartment of the filter with a cotton swab, and the filters were fixed with 8% glutaraldehyde and stained with crystal violet. The extent of cell migration was determined by light microscopy, and within each well, counting was done in nine randomly selected microscopic high power fields.

Flow Cytometric Analysis—Cells were detached by gentle treatment with 0.25% trypsin, 0.05% EDTA in PBS, washed, and incubated for 1 h at 4 °C with antibodies to the {alpha}v{beta}3 (LM609) or {alpha}v{beta}5 integrins (P1F6). Cells were then incubated for1hat4 °C with 10 µg/ml of the secondary antibody, goat anti-mouse IgG conjugated with fluorescein isothiocyanate (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and analyzed at 488 nm on the flow cytometer FACScalibur system (BD Biosciences) equipped with a 5-watt laser.

Binding Assay of {beta}ig-h3—A binding assay was performed as described previously (9). Cells were suspended in medium at a density of 1 x 105 cells/ml, and 1 ml of the cell suspension was preincubated with anti-{alpha}v{beta}3 (LM609) or {alpha}v{beta}5 (P1F6) for 30 min at 37 °C. The cells were incubated with biotinylated {beta}ig-h3 in serum-free media containing 0.1% BSA for 5 h at 4 °C. The cells were then washed three times with phosphate-buffered saline, pH 7.4, before lysis at 4 °C in ice-cold buffer A (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.5% SDS, 0.02% sodium azide, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation at 13,000 rpm for 10 min at 4 °C. Equal amounts of protein were then separated by SDS-PAGE, 8% gel. The amount of biotinylated {beta}ig-h3 was determined by immunoblotting.

To visualize the biotinylated {beta}ig-h3, membranes were incubated with streptavidin conjugated to horseradish peroxidase (Amersham Biosciences). Binding of the peroxidase-labeled antibody was visualized using enhanced chemiluminescence (ECL; Amersham Biosciences). Stripping and reprobing for tubulin (Santa Cruz Biotechnology) immunoblotting as an internal control were performed according to the manufacturer's instructions.

Endothelial Tube Assay—Matrigel (Chemicon) was added (100 µl) to each well of a 96-well plate and allowed to polymerize. Cells were suspended in medium at a density of 3 x 105 cells/ml, and 0.1 ml of the cell suspension was added to each well coated with Matrigel, together with or without the indicated concentrations of each peptide. Cells were incubated for 16–18 h at 37 °C. The cells were then photographed, and tubes were counted and averaged.

Matrigel Plug Assay—An in vivo Matrigel plug assay was performed as described previously (10). Five- to 6-week-old male C57BL/6 mice were used. Matrigel (BD Biosciences) was mixed with 20 units/ml heparin, 150 ng/ml basic fibroblast growth factor (R & D Systems), and synthetic peptide (AnyGen Co. Ltd.). The Matrigel mixture was injected subcutaneously, and after 7 days mice were sacrificed, and the Matrigel plugs were removed and fixed in 4% paraformaldehyde. The plugs were embedded in paraffin, sectioned, and H & E-stained. Sections were examined by light microscopy, and the number of blood vessels from 4 to 6 high power fields (x200) was counted and averaged. Each group consisted of three or four Matrigel plugs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{beta}ig-h3 Mediates HUVEC Adhesion through the {alpha}v{beta}3 Integrin—Recently, we reported that {beta}ig-h3 has two {alpha}3{beta}1 integrin-interacting motifs mediating epithelial cell adhesion (3) and four {alpha}v{beta}5 integrin-interacting motifs mediating fibroblast cell adhesion (4). In the present study, we also found that {beta}ig-h3 mediates endothelial cell adhesion and that each fas-1 domain of {beta}ig-h3 was equally active in mediating endothelial cell adhesion (Fig. 1A). To identify the integrin responsible for endothelial cell adhesion to {beta}ig-h3, we used several integrin function blocking antibodies. As shown in Fig. 1B, endothelial cell adhesion to {beta}ig-h3 was specifically inhibited by antibodies to the {alpha}v{beta}3 integrin and {beta}3 integrin but not by antibodies against other integrins, including the {alpha}v{beta}5, {alpha}3, and {beta}1 integrins. Endothelial cell adhesion to each fas-1 domain was also blocked by a function-blocking antibody to the {alpha}v{beta}3 integrin but not to the {alpha}v{beta}5 integrin (Fig. 1C). To confirm that HUVECs express both the {alpha}v{beta}3 and {alpha}v{beta}5 integrin on their cell surface, we did FACS analysis using specific monoclonal antibodies to both integrins. As shown in Fig. 1D, HUVECs express both integrins, but the expression level of the {alpha}v{beta}5 integrin is far less than the {alpha}v{beta}3 integrin. These results suggest that each fas-1 domain bears a motif mediating endothelial cell adhesion through the {alpha}v{beta}3 integrin.



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FIG. 1.
All fas-1 domains in {beta}ig-h3 mediate HUVEC adhesion by binding to {alpha}v{beta}3 integrin. A, adhesion of HUVEC to each fas-1 domain in {beta}ig-h3. 96-Well plates were coated with {beta}ig-h3-WT or each fas-1 domain overnight at 4 °C. After seeding and incubation, attached cells were quantified by measuring hexosaminidase, as described under ``Experimental Procedures.'' B, identification of integrins mediating the adhesion of HUVEC to {beta}ig-h3. HUVEC were preincubated with the following function-blocking monoclonal antibodies to integrin subunits and then added to the precoated wells: {alpha}3(P1B5), {alpha}5(P1D5), {alpha}v(P3G8), {beta}1(6S6), {beta}3(B3A), {alpha}v{beta}3(LM609), and {alpha}v{beta}5(P1F6). After seeding and incubation, attached cells were quantified by measuring hexosaminidase. C, the effect of the integrin {alpha}v{beta}3 function-blocking antibody on HUVEC adhesion to each fas-1 domain. D, analysis of integrins expressed on the HUVEC surface. Flow cytometric analysis was performed on HUVEC stained with saturating concentration of the following monoclonal antibodies: {alpha}v{beta}3(LM609) and {alpha}v{beta}5(P1F6). The data are expressed as cell number (y axis) plotted as a function of fluorescence intensity (x axis) and are representative of three separate experiments. Negative control cells were incubated with the secondary antibody alone.

 

The YH Motif Is Necessary for {beta}ig-h3-mediated Endothelial Cell Adhesion—To identify the {alpha}v{beta}3 integrin-interacting motif, we used proteins containing deletion mutations in the 4th fas-1 domain, which have been described previously (4). We found that the {alpha}v{beta}3 integrin-interacting motif was present within a fragment corresponding to amino acids 548–614 because this is the smallest fragment that still retains endothelial cell adhesion activity (Fig. 2A). We reported previously (4) that this fragment contains the YH motif, which has been shown to bind the {alpha}v{beta}5 integrin. We suspected that the YH motif may also interact with the {alpha}v{beta}3 integrin to mediate endothelial cell adhesion. We used several substitution mutant 4th fas-1 proteins as described previously (4), whose tyrosine, histidine, and flanking leucine/isoleucine residues were mutated in various combinations (Fig. 2A). Neither mutations on tyrosine and histidine nor mutations on leucine/isoleucine at either side of YH residues abolished cell adhesion activity. On the contrary, any combinatory mutations on YH and flanking leucine/isoleucine significantly reduced cell adhesion activity (Fig. 2A). The results suggest that not only tyrosine and histidine but also flanking leucine/isoleucine residues are required to mediate HUVEC adhesion. To confirm further that the YH motif is responsible for endothelial cell adhesion, we used YH18 synthetic peptides from each fas-1 domain, which have been shown to inhibit the adhesion of fibroblasts to {beta}ig-h3 through the {alpha}v{beta}5 integrin (4), in a cell adhesion assay. These peptides also inhibit endothelial cell adhesion to {beta}ig-h3 in a dose-dependent manner (Fig. 2B). These results suggest that the YH motif is also responsible for mediating endothelial cell adhesion to {beta}ig-h3 through the {alpha}v{beta}3 integrin. Because the YH peptide was reported previously to interact with the {alpha}v{beta}5 integrin of fibroblasts (4) and HUVECs express the {alpha}v{beta}5 integrin, it is expected that the {alpha}v{beta}5 integrin of HUVECs could also be involved in mediating HUVECs adhesion to {beta}ig-h3. To investigate whether {beta}ig-h3-mediated HUVECs adhesion is specifically dependent on the {alpha}v{beta}3 integrin, we further studied the binding affinity of {beta}ig-h3 in the presence of a specific function-blocking antibody to each integrin. Fig. 3A shows that {beta}ig-h3 binds to the HUVECs surface in a dose-dependent manner, and its binding is specifically inhibited by an antibody to the {alpha}v{beta}3 integrin but not by an antibody to the {alpha}v{beta}5 integrin nor IgG (Fig. 3B).



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FIG. 2.
Identification of the {beta}ig-h3 motif mediating HUVEC adhesion. A, effect of mutations in the {beta}ig-h3-domain IV on HUVEC adhesion. {Delta}H1H2(6) is a fragment of domain IV corresponding to amino acids 548–614. Substitution mutations of domain IV are made on tyrosine, histidine, and flanking leucine/isoleucine residues as described under ``Experimental Procedures.'' Mutated amino acids are shown in boldface. Ten µg/ml of indicated proteins were used for coating the plates and incubation with HUVEC. After incubation, cells attached to the surface were quantified. B, dose-dependent inhibition of HUVEC adhesion to {beta}ig-h3 by YH18 synthetic peptides from each fas-1 domain. The sequence of each peptide is shown.

 


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FIG. 3.
The {alpha}v{beta}3 function-blocking antibody inhibits the binding of soluble {beta}ig-h3. A, dose-dependent binding of biotinylated {beta}ig-h3 to HUVECs surface. Cells were incubated with biotinylated {beta}ig-h3 for 5 h at 4 °C. After lysis, the amount of biotinylated {beta}ig-h3 associated with the cells was determined by Western immunoblotting with the streptavidin-conjugated horseradish peroxidase. B, the effect of the integrin {alpha}v{beta}3 function-blocking antibody (Ab) on binding of biotinylated {beta}ig-h3 to HUVECs surface. Cells were preincubated with anti-{alpha}v{beta}3(LM609) or {alpha}v{beta}5(P1F6). The cells were incubated with biotinylated {beta}ig-h3 in serum-free media containing 0.1% BSA for 5 h at 4 °C. After lysis, the amount of biotinylated {beta}ig-h3 associated with the cells was determined by Western immunoblotting with the streptavidin-conjugated horseradish peroxidase.

 

The {alpha}v{beta}3 Integrin Is a Functional Receptor for {beta}ig-h3—To confirm that the {alpha}v{beta}3 integrin mediates endothelial cell adhesion to {beta}ig-h3, we used HEK293 cells stably transfected with a human {beta}3 integrin expression vector. {beta}3/293 cells strongly adhered to {beta}ig-h3, whereas pc/293 cells, which were stably transfected with an empty vector, did not (Fig. 4A). {beta}3/293 cell adhesion to {beta}ig-h3 was specifically inhibited by an antibody to the {alpha}v{beta}3 integrin (Fig. 4B). The YH18 peptides from each fas-1 domain also inhibited {beta}3/293 cell adhesion to the {beta}ig-h3 protein (Fig. 4C). These results confirm that the YH motif of {beta}ig-h3 mediates endothelial cell adhesion through interaction with the {alpha}v{beta}3 integrin.



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FIG. 4.
The {alpha}v{beta}3 integrin is a functional receptor for {beta}ig-h3. A, adhesion to {beta}ig-h3 by HEK293 cells stably transfected with a {beta}3 integrin expression vector. 96-Well plates were coated with BSA or {beta}ig-h3-WT and incubated with the cells, and the cells attached to the surface were quantified. B, the effects of function-blocking integrin antibodies on {beta}3/HEK293 cell adhesion to {beta}ig-h3-WT protein. Cells were preincubated with the following function-blocking monoclonal antibodies specific for integrin subunits and then added to the {beta}ig-h3-WT precoated wells, {alpha}v{beta}3(LM609) and {alpha}v{beta}5(P1F6). After incubation, the cells attached to the substrate were quantified. C, dose-dependent inhibition of {beta}3/HEK293 cell adhesion to {beta}ig-h3 by YH18 synthetic peptides from each fas-1 domain.

 

The YH Motif Mediates Endothelial Cell Migration through the {alpha}v{beta}3 Integrin—We tested whether {beta}ig-h3 mediates endothelial cell migration using a transwell system. We found that the migration of endothelial cells was enhanced in those trans-wells whose undersurface was coated with {beta}ig-h3 and that this effect was inhibited by an antibody to the {alpha}v{beta}3 integrin but not by an antibody to the {alpha}v{beta}5 integrin (Fig. 5, A and B). The YH18 peptide also inhibited endothelial cell migration toward {beta}ig-h3 (Fig. 5, C and D). These results suggest that the YH motif of {beta}ig-h3 mediates endothelial cell migration through the {alpha}v{beta}3 integrin.



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FIG. 5.
The YH motif mediates endothelial cell migration through the {alpha}v{beta}3 integrin. A and B, the effect of the integrin {alpha}v{beta}3 function-blocking antibody on {beta}ig-h3-mediated HUVEC migration. Migration of HUVEC on {beta}ig-h3-WT was assayed using transwell plates. Cells were preincubated with anti-{alpha}v{beta}3 monoclonal antibody (LM609) and anti-{alpha}v{beta}5 monoclonal antibody (P1F6) for 30 min before adding the cells into the upper wells of the transwell plate. C and D, the effect of the YH18 peptide on {beta}ig-h3-mediated HUVEC migration. Cells were preincubated with the YH18 peptide for 30 min before adding the cells into the upper wells of the transwell plate. Cells that migrated through the filter and into the lower side were fixed and stained. Cell migration was quantified by counting migrated cells in nine microscope fields. con., control.

 

YH18 Peptides Inhibit Angiogenesis in Vitro and in Vivo—In the next experiment, we tested whether the YH18 peptide can inhibit angiogenesis in vitro. The YH18 peptide selectively inhibited endothelial tube formation on Matrigel in a dose-dependent manner (Fig. 6, A and B), whereas the control peptide did not. A half-inhibition was observed at 500 µM. To test the in vivo effect of the YH18 peptide, we performed a Matrigel plug assay in mice. Matrigel was placed in the presence of bFGF with or without increasing concentrations of the YH18 peptide. A significant reduction in the number of blood vessels was observed at 500 µM (Fig. 7, A and C). These results suggest that the YH peptide inhibits angiogenesis in both in vitro and in vivo assays at a similar concentration.



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FIG. 6.
The YH18 peptide inhibits HUVEC tube formation in vitro. A and B, cells were preincubated with or without YH18 (500 µM or 1 mM) and then seeded in 96-well plates coated with Matrigel. After 16–18 h of culture, the number of tube branches in a low power field was counted (three independent wells were counted and averaged). con., control.

 


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FIG. 7.
YH18 peptides inhibit angiogenesis in vivo. A, the YH18 (500 µM or 1 mM) peptide was mixed with Matrigel plus and then injected into the flank of a mouse. After 7 days, the animals were sacrificed, and the plugs were removed and scanned at high resolution. B and C, sections of each Matrigel plug stained by hematoxylin and eosin were examined by light microscopy (x200 magnification), and the number of blood vessels from 4 to 7 high power fields was counted and averaged. Inset in B, high magnification view (x400) of blood vessels. Arrow indicates the position magnified in insets. Arrowheads indicate the blood vessels. con., control.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{beta}ig-h3 has been known to mediate adhesion of several cell types, including epithelial cells (3, 7), fibroblasts (4), chondrocytes (11), and vascular smooth cells (12). We reported that {beta}ig-h3 has two short motifs interacting with the {alpha}3{beta}1 integrin for mediating adhesion of corneal epithelial cells (3) and keratinocytes (7). Recently, we identified new motifs in the fas-1 domains of {beta}ig-h3 that mediate fibroblast adhesion (4). This motif was designated the YH motif because it contains highly conserved tyrosine and histidine residues. In addition, the YH motif has a few more conserved leucines and isoleucines flanking the tyrosine and histidine residues. At least 18 amino acids, including these conserved residues, are required to mediate fibroblast adhesion. The YH motif mediates the adhesion of fibroblasts by interacting with the {alpha}v{beta}5 integrin. In the present study, we first demonstrated that {beta}ig-h3 also mediates adhesion and migration of endothelial cells acting through the {alpha}v{beta}3 integrin. Interestingly, the {alpha}v{beta}3 integrin-interacting motifs turned out to be the YH motif. The ability of the YH peptide to interact with both the {alpha}v{beta}3 and {alpha}v{beta}5 integrins is not surprising because the {alpha}v{beta}3 and {alpha}v{beta}5 integrins are known to share ligands (13), and both bind to the RGD peptide and its peptidomimetics (14). Because we found that the interaction of the YH peptide with the {alpha}v{beta}5 (4) and {alpha}v{beta}3 integrins (data not shown) are RGD-dependent, we hypothesize that the YH and RGD peptides interact with the {alpha}v{beta}3 and {alpha}v{beta}5 integrins in a similar manner. Because the YH peptide is much larger than the RGD peptide, their binding sites in each of the {alpha}v{beta}3 and {alpha}v{beta}5 integrins could be different. Alternatively, their binding sites may overlap or the binding of one peptide might cause a conformational change that affects the binding of the other peptide. If the YH motif interacts with both the {alpha}v{beta}3 and {alpha}v{beta}5 integrins, why are the {alpha}v{beta}5 integrins of HUVECs not involved in {beta}ig-h3-mediated adhesion? Our FACS analysis shows that the amount of the {alpha}v{beta}5 integrin of HUVECs is much lower than that of the {alpha}v{beta}3 integrin. Therefore, the {alpha}v{beta}3 integrin may function predominantly in mediating HUVECs adhesion to {beta}ig-h3. Alternatively, the YH motif may have different binding affinity with the {alpha}v{beta}3 and {alpha}v{beta}5 integrins, or its binding affinity depends on the activation state of the {alpha}v{beta}3 and {alpha}v{beta}5 integrins. Our previous mutational study shows mutations on either YH residues or leucine/isoleucine residues of either side of YH partially inhibit an activity of {beta}ig-h3 in mediating fibroblasts adhesion (4), whereas in the present study, they did not significantly affect an activity of {beta}ig-h3 in mediating HUVECs. It suggests that amino acid residues of YH motif may be required to interact differently with each of the {alpha}v{beta}3 and {alpha}v{beta}5 integrins. It is well known that RGD peptides in different contexts or constraining their conformation with cross-linkers show large differences in their affinity for different integrins (1518). However, it remains to be further studied how the YH motif interacts differently with the {alpha}v{beta}3 and {alpha}v{beta}5 integrins and how each amino acid residue such as tyrosine, histidine, and leucine/isoleucine affects the structure and activity of the YH motif.

The {alpha}v{beta}3 integrin and its closely related {alpha}v{beta}5 integrin have been known to play a role in angiogenesis (19). Thus, numerous monoclonal antibodies and many RGD mimetics have been tested for their ability to block angiogenesis by inhibiting the function of these integrins. It is still unclear whether the {alpha}v{beta}3 and {alpha}v{beta}5 integrins are proangiogenic or not (20). In fact, recently, these two integrins were hypothesized to be negative regulators of angiogenesis (20). Nevertheless, many molecules targeting these integrins have been reported to have anti-angiogenic effects. Several groups suggested that the {alpha}v{beta}3 integrin is a receptor for various proteolytic fragments of extracellular matrix proteins that can act as anti-angiogenic factors (2123). For example, a fragment of the type IV collagen {alpha}3 chain acts through the {alpha}v{beta}3 integrin to induce apoptosis of endothelial cells, thereby inhibiting angiogenesis (10). Based on findings that the YH peptide interacts with the {alpha}v{beta}3 integrin, we believed that the YH peptide may act through the {alpha}v{beta}3 integrin of endothelial cells to inhibit angiogenesis. We found that the YH peptide inhibited endothelial cell adhesion and migration. Because angiogenesis depends on specific endothelial cell adhesion and migration processes mediated by the {alpha}v{beta}3 integrin (24, 25), the YH peptide may disrupt the interaction of endothelial cells with substrates such as fibronectin and vitronectin. In vitro angiogenesis assays using Matrigel showed that the YH peptide significantly reduces the number of capillary tubes formed. This anti-angiogenic effect was further confirmed by an in vivo experiment in C57BL/6 mice using Matrigel plugs demonstrating that the YH peptide effectively inhibits bFGF-induced neovascularization. The anti-angiogenic activity of the YH peptide does not seem to be associated with cell proliferation because the YH peptide does not significantly affect endothelial cell growth in the presence of serum or growth factors such as bFGF and VEGF at a concentration of 500 µM (data not shown), which clearly shows anti-angiogenic activity in both in vitro and in vivo assays.

We reported previously (4) that the YH motif consisting of 18 amino acids is minimally required to mediate cell adhesion and hence is less active than the fas-1 domain fragment in inhibiting cell adhesion to {beta}ig-h3. Expectedly, the ability of the YH peptide to inhibit endothelial cell adhesion and migration is less than that of the 4th fas-1 domain of {beta}ig-h3 (data not shown). Accordingly, its anti-angiogenic activity is weaker than that of the fas-1 domain fragment (data not shown). For this reason, we are currently using the fas-1 domain protein rather than the YH peptide in in vivo experiments to test its anti-tumor effects.

The YH motif is highly conserved in many fas-1 domains of several proteins. Recently, the human protein FEEL-1 (stabilin-1), which has seven fas-1 domains and is expressed in endothelial cells, was suggested to play a role in angiogenesis (26). Although there is no direct in vivo evidence showing that the fas-1 domain-containing proteins are involved in angiogenesis, our results together with the above report suggest that the fas-1 domain-containing proteins or their degradation products may regulate angiogenesis and therefore could be used in the development of drugs targeting angiogenesis.


    FOOTNOTES
 
* This work was supported by National Research Laboratory Program M10104 [GenBank] 00036-01J0000-01610. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biochemistry, Kyungpook National University School of Medicine, 101 Dongin-dong, Jung-gu, Taegu, 700-422, Korea. Tel.: 82-53-420-6933; Fax: 82-53-422-1466; E-mail: iskim{at}knu.ac.kr.

1 The abbreviations used are: HUVECs, human umbilical vein endothelial cells; FACS, fluorescence-activated cell sorter; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HEK, human embryonic kidney; WT, wild type; bFGF, basic fibroblast growth factor. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Jeffrey Smith (Burnham Institute, San Diego) for providing the {beta}3/293 cells.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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