Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:360-369

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Klein-Soyer, C.
Right arrow Articles by Lanza, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Klein-Soyer, C.
Right arrow Articles by Lanza, F.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Injuries
*Wounds
Hazardous Substances DB
*L-TYROSINE
Related Collections
Right arrow Endothelium/vascular type/nitric oxide
Right arrow Other Vascular biology
Right arrow Angiogenesis
Right arrow Cell biology/structural biology
(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:360.)
© 2000 American Heart Association, Inc.

Vascular Biology

CD9 Participates in Endothelial Cell Migration During In Vitro Wound Repair

Presented in part at the XIVth Congress of the International Society on Thrombosis and Haemostasis, June 6–12, 1997, Florence, Italy, and published in abstract form (Thromb Haemost. 1997;[suppl]:138).

Claudine Klein-Soyer; David O. Azorsa; Jean-Pierre Cazenave; François Lanza

From INSERM U. 311, Etablissement de Transfusion Sanguine de Strasbourg (C.K.-S., J.-P.C., F.L.), Strasbourg, France, and the Laboratory of Cancer Genetics (D.O.A.), National Institutes of Health/National Human Genome Research Institute, Bethesda, Md.

Correspondence to Dr Claudine Klein-Soyer, Etablissement de Transfusion Sanguine, INSERM U.311, 10 rue Spielmann, B.P. 36, Strasbourg Cédex, France. E-mail claudine.soyer{at}etss.u-strasbg.fr


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—CD9, a widely expressed membrane protein of the tetraspanin family, has been implicated in diverse functions, such as signal transduction, cell adhesion, and cell motility. We tested the effects of an anti-CD9 monoclonal antibody (ALMA.1) on the migration and proliferation of human vascular endothelial cells (ECs) during repair of an in vitro mechanical wound mimicking angiogenic processes. ALMA.1 induced dose-dependent inhibition of wound repair with a 35±1.5% decrease at 20 µg/mL. Only cell migration was affected, because the rate of proliferation of ECs at the lesion margin was not modified and because the inhibition of repair was also observed for nonproliferating irradiated ECs. Monoclonal antibodies against CD63 tetraspanin (H5C6) and control mouse IgG (MOPC-21) were inactive. CD9, one of the most abundant proteins at the surface of ECs, colocalized with ß1 or ß3 integrins on EC membranes in double-labeling immunofluorescence experiments with ALMA.1 and an anti-ß1 (4B4) or anti-ß3 (SDF.3) monoclonal antibody. Moreover, ALMA.1 and 4B4 had additive inhibitory effects on lesion repair, whereas 4B4 alone also inhibited EC proliferation. In transmembrane Boyden-type assays, ALMA.1 induced dose-dependent inhibition of EC migration toward fibronectin and vitronectin with 45±6% and 31±10% inhibition, respectively, at 100 µg/mL. 4B4 inhibited migration toward fibronectin at 10 µg/mL but had no effect in the case of vitronectin. Adhesion of ECs to immobilized anti-CD9 monoclonal antibodies induced tyrosine-phosphorylated protein levels similar to those observed during interactions with ß1 or ß3 integrins. These results point to the involvement of CD9 in EC adhesion and migration during lesion repair and angiogenesis, probably through cooperation with integrins. As such, CD9 is a potential target to inhibit angiogenesis in metastatic and atherosclerotic processes.


Key Words: CD9 • integrins • endothelial cells • cell migration • wound repair


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
CD9 is a widely expressed transmembrane protein of the tetraspanin superfamily (also known as TM4SF) with a broad tissue distribution and regulated expression depending on the embryonic or developmental stage.1 2 CD9 is found in various tumor cells,3 4 in neural tissue,5 and in the vasculature in endothelial cells (ECs),6 smooth muscle cells,7 and platelets.8 Transfection experiments and the development of specific monoclonal antibodies (mAbs) have contributed to define possible functions for CD9. First, CD9 has been implicated in motility and tumor processes.9 10 Overexpression in adenocarcinoma cells suppresses the motility and metastatic potential,10 CD9 expression in breast and pulmonary cancer is inversely related to the metastatic stage and to patient survival,4 and anti-CD9 mAbs inhibit the motility of various leukemic cell lines11 but enhance Schwann cell migration.5 Second, CD9 has been found to participate in cell adhesion and aggregation. Anti-CD9 mAbs increase neutrophil adherence to endothelium12 and adhesion of lymphocyte progenitors to bone marrow stromal fibroblasts13 and induce the aggregation of strongly CD9-positive leukemic cell lines,11 whereas immobilized anti-CD9 mAbs promote the adhesion of Schwann cells.14 Finally, CD9 has been implicated in signal transduction.15 16 However, because of their short intracytoplasmic domains, it would seem unlikely that intracellular signaling can occur through TM4SF molecules alone. The wide diversity of functions attributed to tetraspanins is probably related to the association of these molecules with other protein complexes.

Coimmunoprecipitation and cocapping experiments have demonstrated that some tetraspanins facilitate the association of coreceptor molecules, such as CD4 and CD8 on T cells, and that some tetraspanins form tetraspanin complexes, which can in turn associate with coreceptors or integrins. Thus, CD9 forms complexes with CD81 or CD63, which associate with {alpha}vß1 or {alpha}6ß1 integrins (reviewed in Reference 11 ). In platelets, where anti-CD9 antibodies trigger aggregation, CD9 is associated with the integrin {alpha}IIbß3.17 18 19 Similarly, CD9 associates with ß1 integrins in various leukemic cell lines and inhibits their transmembrane migration.11 Another member of TM4SF, CD63, which has been identified on activated platelets20 and is a component of Weibel-Palade bodies in ECs,21 participates in adhesion processes and also forms specific complexes with some ß1 integrins in human fibrosarcoma and erythroleukemia cell lines.22

Several years ago, we developed a model of a mechanical wound in confluent ECs that allows the concomitant study of cell migration and proliferation during the repair process and mimics mechanisms encountered in angiogenesis.23 24 In view of the crucial role of angiogenesis in tumor progression and metastasis25 and the relevance of integrins and their association with tetraspanins in these processes,26 27 we tested in the present study the effects of anti-tetraspanin antibodies on the migration and proliferation of human vascular ECs during lesion repair. We also examined the effects of these antibodies on transmembrane migration toward matrix proteins in a modified Boyden chamber assay. Finally, we investigated the possible colocalization of CD9 with ß1 and ß3 integrins in ECs.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
Cell culture medium (medium 199 with Hanks’ salts/RPMI 1640), HEPES, PBS with Dulbecco’s formulation, trypsin-EDTA, L-glutamine, antibiotics (penicillin and streptomycin), and fungizone were from GIBCO. Tissue-culture Petri dishes (35-mm diameter) were from Corning Glass Works; 24- and 96-multiwell plates and cell-culture insert migration chambers, from Falcon, Becton-Dickinson Co; and Laboratory-Tek tissue-culture chamber slides, from Nunc, Inc. Pooled heat-inactivated human serum (HS), hepatitis B, and HIV-free and HS albumin (HSA) were from the Etablissement de Transfusion Sanguine de Strasbourg. Human fibronectin (FN) and vitronectin (VN) were purified from human plasma as previously described.28 29 Anti-CD9 mAbs (clone ALMA.1, IgG1, {kappa}; clone ALMA.3, IgG2a, {kappa}; and clone RPM.13, IgG1, {kappa}), anti-CD81 mAb (clone Z81.1, IgG1, {kappa}), and anti-ß3 mAb (clone SDF.3, IgG2a, {kappa}) were produced by a previously described procedure in mice immunized with washed human platelets. The hybridoma culture supernatants were screened for binding to recombinant GST-CD9 fusion protein by ELISA30 and for binding to washed platelets by flow cytometry or immunoprecipitation of biotin-labeled platelet protein.31 Hybridomas secreting anti-CD9 or anti-ß3 mAbs were subcloned twice. Anti-CD9 F(ab')2 fragments were prepared as follows: Purified ALMA.1 IgG (3 mg) was digested overnight at 37°C on a rocking platform with 400 µg pepsin (Boehringer-Mannheim) in 0.1 mol/L sodium acetate and 0.15 mol/L NaCl, pH 4.5. After adjusting the pH to 7 with 1 mol/L Tris, digestion was checked by SDS-PAGE analysis. The digest was dialyzed against 3 changes of PBS buffer, pH 7.2, and applied to a Superdex 200 HR 10/30 column (Pharmacia Biotechnology) equilibrated in the same buffer. The F(ab')2-containing fraction still contaminated with intact IgG was finally purified on an ion-exchange Mono Q-HR column (Pharmacia) by applying a linear 0 to 300 mmol/L NaCl gradient in 50 mmol/L Tris buffer, pH 8. The anti-CD63 mAb (clone H5C6, IgG1, {kappa}) has been described elsewhere,32 33 the anti-ß1 mAb (clone 4B4, IgG1) was from Coulter (Coultronics), and the mAb to the Fc{gamma} RII receptor (clone IV.3, IgG2b) (MOPC-21) has also been described previously.34 Purified mouse IgG1 ({kappa} chain) control immunoglobulins, biotinylated anti-phosphotyrosine mAbs (clone PT 66), and Arg-Gly-Asp-Ser (RGDS) peptide were purchased from Sigma Immuno Chemicals (Sigma-Aldrich Corp), and horseradish peroxidase–conjugated streptavidin was from Pierce Chemical Co (Pierce Europe, B.V.). Sheep anti-bromodeoxyuridine (anti-BrdU) polyclonal antibodies were from Fitzgerald. Purified mouse IgG2a ({kappa} chain) (MOPC-173) control immunoglobulins were obtained from Pharminogen, Becton Dickinson Co. Normal donkey serum, normal goat serum, affinity-purified peroxidase-conjugated donkey anti-sheep IgG (minimal cross-reactivity to human proteins), normal mouse serum, Cy2- and Cy3-affiniPure goat anti-mouse IgG (GAM; minimal cross-reactivity to human, bovine, and horse serum proteins) and FITC-labeled GAM were from Jackson Immunoresearch Laboratories. Mowiol 4–88 was obtained from Calbiochem-Novabiochem Corp. When it was required, the mAbs were dialyzed against PBS and filter-sterilized. Irradiation of cells was performed in an irradiator for blood products (model IBL437 C, Commissariat à l’Energie Atomique) with a 137Cs radiation source delivering 10 Gy/73 s under water. Electrophoresis supplies were obtained from Bio-Rad Laboratories, Immobilon-P transfer membranes were from Millipore Corp, the enhanced chemiluminescence detection kit was from Amersham, and cellulose polyacetate paper (Sepraphore III, No. 51003) was from Gelmann. All other chemicals were of analytical grade and were from Sigma or Merck.

Cell Culture
Human saphenous vein or mammary artery ECs were collected from vessel fragments obtained during coronary bypass surgery and cultured as previously described.35 36 After they were thawed, the cells were seeded on purified human FN in culture dishes and used from the second to the fifth passages. The culture medium was medium 199/RPMI 1640 (50/50) containing 10 mmol/L HEPES, 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL fungizone, and 30% pooled heat-inactivated HS. Experiments performed with ECs from saphenous veins or mammary arteries gave similar results.

Adhesion Assay
In a first set of experiments, cell adhesion was determined for longer times under the usual optimized culture conditions. Trypsinized ECs were preincubated with antibodies for 30 minutes at room temperature under constant gentle shaking and seeded at a concentration of 5000 cells per square centimeter in medium containing 5% HS on FN- or VN-coated (7.2 to 10 µg per well) 96-multiwell plates. After 24 hours, nonadherent cells were eliminated by extensive rinsing with PBS. Adherent ECs were fixed at room temperature in 2% paraformaldehyde (PF) solution for 20 minutes, stained with May-Grünwald Giemsa stain, and counted with the use of an inverted microscope (Nikon Diaphot-TMD) at x100 magnification. In further experiments, cell adhesion was measured for shorter times in the absence of serum. The concentration of adhesive protein was lowered to 0.5 µg per well, nonsaturated sites were blocked with 5% HSA, HS was replaced by 1% HSA, and inhibitory substances or antibodies were added to the wells immediately before EC seeding. The duration of the assays was limited to 30 minutes, and at the end of this period, the multiwell plates were processed as described above.

Proliferation Assay
ECs were seeded at a concentration of 5000 cells per square centimeter on FN-coated 96-multiwell plates in medium containing 30% HS. On days 1 and 4 after seeding, the medium was removed and replaced with fresh medium containing 10% HS and antibodies. At day 7, adherent ECs were fixed in 2% PF solution, and EC proliferation was estimated by quantification of crystal violet staining of the cells37 with a microplate reader (Molecular Devices).

Mechanical Injury of Confluent ECs and Lesion Repair
The procedure for establishment of a mechanical lesion of confluent ECs in vitro and analysis of lesion repair has been described in earlier publications.38 39 Briefly, a 6-mm-diameter endothelial monolayer was detached from the underlying extracellular matrix with a disk of polyacetate paper. This step constituted time zero of the experiment. At this time, serum concentration in the medium was reduced to 5%, and antibodies were added. Antibodies were further added over a concentration range of 0.1 to 50 µg/mL during the repair process, and nonimmune mouse IgG was used for the control. After an 18-hour pulse with 10 µmol/L BrdU, culture dishes were removed on day 2 and fixed in 2% PF solution as described above. In some experiments, to hinder the capacity of ECs to proliferate, culture dishes were irradiated before lesion development with 10 Gy, a dose that inhibits [3H]thymidine incorporation by 80% to 90%.23 Proliferating ECs were visualized by BrdU incorporation into the nuclei40 by use of sheep anti-BrdU and peroxidase-conjugated donkey anti-sheep antibodies. Replicating cells displayed black nuclei after 3,3'-diaminobenzidine staining, whereas nonreplicating cells had light purple nuclei after Giemsa dye counterstaining.

Cell Migration Assay
Transmembrane cell migration assays41 42 were performed in modified Boyden chambers by use of cell culture inserts composed of a porous 8-µm (1x105 pores per square centimeter) polyethyleneterephthalate membrane with a diameter of 6.25 mm and a thickness of 11 µm. Soluble adhesive proteins (5 µg/mL in 600 µL of serum-free culture medium) were placed in the lower compartment (24-multiwell plate) and incubated at 37°C for 30 minutes before cell addition. Nonspecific random migration was determined by using HSA (5 µg/mL) instead of adhesive proteins. ECs (105 cells in 100 µL), preincubated with antibodies for 20 minutes at room temperature under gentle shaking, were added to the upper cell culture insert, and cell migration was measured after 6 hours of incubation at 37°C. Nonmigrating cells were removed from the upper surface of the insert with a cotton swab, whereas those that had migrated to the lower face of the membrane were fixed with 0.1% crystal violet in 0.1 mol/L sodium borate, pH 9.0, containing 2% ethanol. EC counts were performed as described above at x200 magnification.

Detection of Protein Tyrosine Phosphorylation
Protein tyrosine phosphorylation was explored in different situations: (1) in adherent confluent ECs, (2) in ECs incubated in suspension with antibodies before seeding, and (3) in ECs adherent to adsorbed antibodies or adhesive proteins for a short time (30 minutes). The corresponding assays were performed as follows: (1) Confluent ECs in 35-mm-diameter Petri dishes were incubated in medium containing 5% HS and exposed for various time periods (15 minutes to 48 hours) at 37°C to anti-CD9 mAbs (50 µg/mL), in the presence or absence of the phosphatase inhibitor sodium orthovanadate (50 µmol/L). (2) Confluent ECs were trypsinized and seeded (2x105 cells per well) on FN (0.5 µg per well)–coated 24-multiwell plates. Antibodies (ALMA.1, 4B4, and SDF.3) at 50 µg/mL or 1% HSA (control) were preincubated with the cells for 30 minutes at room temperature or added just before seeding, and ECs were allowed to adhere for 30 minutes at 37°C. (3) Trypsinized ECs were seeded (2x105 cells per well) on antibody-coated 24-multiwell plates. The wells were first incubated with GAM (10 µg/mL) overnight at 4°C and blocked with 5% HSA for 1 hour at 37°C. After they were rinsed with PBS, the wells were incubated with the antibodies (MOPC-21, MOPC-173, ALMA.1, 4B4, and SDF.3) at 10 µg/mL for 2 hours at 37°C, and ECs were then added and allowed to adhere for 30 minutes at 37°C. At the end of the adhesion period, ECs from duplicate wells were fixed and stained as described in Mechanical Injury of Confluent ECs and Lesion Repair.

On completion of the assays (1 to 3), the cells were rinsed with PBS and solubilized in 500 µL lysis buffer (40 mmol/L Tris, pH 6.8, 10% ß-mercaptoethanol, 2% SDS, 10% glycerol, and 0.2% bromophenol blue) containing 1 mmol/L sodium orthovanadate, 5 µg/mL 4-amidinophenylmethanesulfonyl fluoride, 5 µg/mL leupeptin, and 8 µg/mL aprotinin. The lysates were stored at -20°C until gel electrophoresis. Nuclei were removed by microcentrifugation, and after SDS-PAGE separation on 7.5% gels, the proteins were electroblotted onto Immobilon-P transfer membranes. The blots were then incubated for 2 hours at room temperature with biotinylated anti-phosphotyrosine mAbs, incubated for a further 2 hours with horseradish peroxidase–conjugated streptavidin, and developed by use of an enhanced chemiluminescent detection kit according to the manufacturer’s instructions.

FACS Analyses
ECs grown to 70% or 100% confluence were washed extensively with PBS. Adherent cells were removed by incubation in PBS containing 5 mmol/L EDTA at 4°C for 10 minutes, by scraping the cells, or by quick enzymatic detachment (2 minutes, 37°C) in trypsin-EDTA solution (0.05%/0.02%) followed by enzyme inactivation in medium containing 30% HS. All 3 procedures gave similar results in fluorescence-activated cell sorting (FACS) analyses. The washed cells were resuspended at 106 cells per milliliter in RPMI medium containing 5% normal goat serum and 0.1% sodium azide, and 105 cells were incubated with the primary antibody (10 µg/mL) for 1 hour on ice. After they were washed with PBS, the cells were incubated with FITC-labeled GAM (10 µg/mL) for 1 hour on ice and analyzed with a cell sorter (FACScalibur, Becton Dickinson). Gates were set for analysis of intact cells only.

Double-Labeling Immunofluorescence
ECs grown on chamber slides were washed in PBS, fixed in 2% PF solution at room temperature, and then permeabilized and blocked in PBS containing 0.1% saponin and 1% BSA at 4°C overnight. The slides were incubated with a first set of primary antibodies (ALMA.1, SDF.3, 4B4, H5C6, MOPC-173, and MOPC-21) at 10 µg/mL in PBS containing 0.1% saponin, 3% BSA, and 1% normal goat serum (PBS-SBN) for 1 hour; they were then washed in PBS and incubated with Cy2- or Cy3-conjugated GAM in PBS-SBN for 1 hour. Free sites were blocked with 1% normal mouse serum in PBS. After they were washed, the cells were incubated with a second set of primary antibodies (ALMA.1, SDF.3, 4B4, H5C6, MOPC-173, and MOPC-21) at 10 µg/mL for 1 hour in PBS-SBN, washed again, and incubated with Cy2- or Cy3-conjugated GAM in PBS-SBN for 1 hour. The slides were then rewashed, mounted in Mowiol 4–88 solution with coverslips, and examined under a fluorescence microscope (Leica, DMLD). Single- or double-labeled isotype controls visualized with Cy2- or Cy3-conjugated GAM were negative. In addition, labeling of specific primary antibodies with either Cy2- or Cy3-conjugated GAM in the first or second position did not modify their labeling pattern (data not shown).

Image Analysis
The repair process was followed quantitatively by image analysis with Visiolab 1000 software (Biocom) as previously described.24 39 After measurement of the lesion area, cell density was calculated by counting the nuclei in 4 random 0.25-mm2 calibrated fields at the leading edge of the lesion, in duplicate samples for each condition. This method is independent of cell shape and size. The proliferating cells, visualized by BrdU incorporation, were expressed as the percentage of the cell density in the corresponding field. To facilitate the comparison of identical conditions in separate experiments, results were expressed as the percentage of the control value set at 100%.

Statistical Analyses
The effects of the various antibodies on lesion repair and EC adhesion, migration, and proliferation were compared by ANOVA followed by the Newman-Keuls test with the use of the statistical software STAT-ITCF (ITCF).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Expression of CD9, CD63, CD81, ß1, ß3, and Fc{gamma}RII on ECs
Surface expression of CD9, CD63, and CD81 tetraspanins, ß1 and ß3 integrins, and Fc{gamma}RII on ECs was examined by flow cytometry (Figure 1Down). The widely expressed tetraspanins CD9 and CD81 and ß1 integrin were strongly expressed at the surface of freshly trypsinized ECs. CD63 and ß3 integrin displayed levels of expression {approx}10 times lower, whereas no surface expression of Fc{gamma}RII (CD32) could be detected on ECs. Confluent and proliferating ECs exhibited comparable levels of expression of the different proteins tested (data not shown). In subsequent experiments, ALMA.1 was used as the anti-CD9 mAb.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 1. FACS analysis of CD9 (mAbs ALMA.1, ALMA.3, and RPM.13), ß1 (mAb 4B4), ß3 (mAb SDF.3), CD81 (mAb Z81.1), CD63 (mAb H5C6), and Fc{gamma}RII receptor (mAb IV.3) expression on ECs. Trypsinized ECs were stained with monoclonal antibodies against tetraspanins, integrins, and the Fc{gamma}RII receptor, followed by a FITC-conjugated second antibody, and analyzed on a FACScalibur as described in Methods. The fluorescence histograms of stained cells (thick line) were compared with those of isotype controls (thin line).

Effects of Anti-CD9, -CD63, -ß1, and -ß3 Antibodies on EC Lesion Repair
Anti-CD9 (ALMA.1) and anti-ß1 and anti-ß3 antibodies tested in the range 0.1 to 50 µg/mL induced dose-dependent inhibition of lesion repair with decreases of 35±5%, 41±3%, and 27±2.5%, respectively, at 50 µg/mL (P<0.05) (Figure 2ADown). The percentage of proliferating cells at the lesion margin (see Methods) was not affected by anti-CD9 or anti-ß3, suggesting that these antibodies did not act by inhibiting cell proliferation, whereas anti-ß1 reduced the percentage of proliferating cells by 28±3% at 20 µg/mL (P<0.05). Anti-CD63 had no effect on lesion repair (Figure 2ADown). Identical results were obtained when anti-CD9 F(ab)'2 fragments were used instead of intact IgG (data not shown) or anti-CD9 from the clone ALMA.3, whereas antibodies from the clone RPM.13 against rat CD9 were inefficient. Low concentrations of anti-CD9 (1 µg/mL) and anti-ß1 (1 µg/mL) antibodies, each of which when added separately induced moderate but significant inhibition, had additive inhibitory effects on lesion repair, although the rate of cell proliferation was identical to the control rate (Figure 2BDown). To confirm the effect of the anti-CD9 antibody on cell migration, EC proliferation was blocked with a 10-Gy dose of radiation before establishing the lesion. As expected, anti-CD9 inhibited the lesion repair of irradiated ECs to the same extent (28%) as that of nonirradiated ECs (data not shown).



View larger version (34K):
[in this window]
[in a new window]
  		Figure 2. Effects of anti-CD9 (ALMA.1), anti-CD63 (H5C6), anti-ß1 (4B4), and anti-ß3 (SDF.3) antibodies on the lesion repair of confluent ECs. A, Increasing concentrations of the antibodies were added during the repair process, and the lesion repair (•) and percentage of proliferating cells at the lesion margin ({circ}), determined as described in Methods, were expressed as the percentage of control values. Results are the mean±SEM of 2 or 3 separate experiments with 2 dishes per experimental condition. B, Effects of a low concentration of anti-CD9 and/or anti-ß1 on the lesion repair are shown. Anti-CD9 and anti-ß1 antibodies were added separately or together, at a concentration of 1 µg/mL, during the repair process. The repaired area and rate of cell proliferation at the lesion margin were determined, and results are the mean of 2 separate experiments with 2 dishes per experimental condition. *P<0.05 vs control values; **P<0.05 vs separately added anti-CD9 or anti-ß1 values.

Effect of Anti-CD9 Antibodies on EC Proliferation
Sparsely seeded ECs were allowed to proliferate on FN in the presence of antibodies (0.1 to 50 µg/mL) for 7 days. Compared with control values, neither anti-CD63, anti-ß3, nor control IgG significantly affected EC proliferation at any of the concentrations tested, whereas anti-ß1 antibodies induced dose-dependent inhibition, reaching a plateau value of 28±11% (P<0.05) at 10 µg/mL (Figure 3Down). In contrast, anti-CD9 induced a significant increase in EC proliferation, with a mean of 40±4% at 20 µg/mL.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. EC proliferation in the presence of anti-CD9 (ALMA.1), anti-CD63 (H5C6), anti-ß1 (4B4), and anti-ß3 (SDF.3) antibodies. Increasing concentrations of antibodies were added during the proliferation of sparsely seeded ECs. Cell proliferation was estimated on day 7 by quantification of crystal violet staining as described in Methods and expressed as the percentage of ECs proliferating in the absence of antibodies set at 100%. OD indicates optical density. Results are the mean±SEM of 2 to 4 separate experiments with triplicate assays for each condition. *P<0.05 vs control values.

Effect of Anti-CD9 Antibodies on EC Adhesion
Adhesion to FN- or VN-coated culture dishes under cell culture conditions was not modified in ECs preincubated with anti-CD9 antibodies (1 to 50 µg/mL) for 30 minutes compared with ECs adhering in the absence of antibodies or after incubation with control IgG. Anti-ß1 was likewise inactive under these conditions (data not shown). When adhesion assays were performed for shorter incubation times in the absence of serum, anti-CD9 did not inhibit cell adhesion to either FN or VN but enhanced it by 19±3% (P<0.05, Figure 4Down). The controls, RGDS peptide (1 to 5 mmol/L), and EDTA (1 to 2 mmol/L) induced dose-dependent inhibition of EC adhesion to both FN and VN. In the same conditions, the anti-ß1 antibody 4B4 inhibited EC adhesion to FN slightly but significantly by 21±6% and 21±11% (P<0.05) at 10 and 20 µg/mL, respectively, thus confirming the blocking capacity of this antibody,43 which did not, however, inhibit adhesion to VN (Figure 4Down).



View larger version (61K):
[in this window]
[in a new window]
  		Figure 4. Effects of anti-CD9 (ALMA.1) and anti-ß1 (4B4) antibodies on EC adhesion. Trypsinized ECs were incubated with increasing concentrations of anti-CD9 and anti-ß1 antibodies and allowed to adhere to FN or VN as described in Methods. As controls, ECs were seeded under identical conditions in the presence of mouse IgG, RGDS peptide, or EDTA. Adherent ECs were counted after staining of the cells with May-Grünwald Giemsa dye, and adhesion was expressed as the percentage of the adhesion of untreated control cells. Results are the mean±SEM of 2 to 7 separate experiments performed in triplicate. *P<0.05 vs control values.

Anti-CD9 Antibodies Inhibit EC Transmembrane Migration Toward FN or VN
In Boyden chamber experiments, dose-dependent inhibition of migration of the cells toward FN or VN was induced in ECs incubated with anti-CD9 antibodies (ALMA.1) at 0.01 to 100 µg/mL compared with ECs migrating in the absence of antibodies (Figure 5Down), with maximal effects of 45±6% for FN and 31±10% for VN (P<0.05). Anti-CD9 from the clone ALMA.3 was inefficient in transmembrane experiments. Anti-ß1 antibodies tested in the same concentration range induced 44±7% inhibition of EC migration toward FN at 10 µg/mL (P<0.05). In contrast, higher concentrations of anti-ß1 no longer inhibited cell migration toward FN, whereas the same antibody had only little effect on migration toward VN at any concentration. Anti-CD63 antibodies (100 µg/mL) did not affect EC migration toward FN or VN, and nonspecific migration determined with the use of HSA represented <1%.



View larger version (59K):
[in this window]
[in a new window]
  		Figure 5. Modulation of transmembrane EC migration toward FN or VN by anti-CD9 (ALMA.1) and anti-ß1 (4B4) antibodies. FN or VN (5 µg/mL) in serum-free medium was placed in the lower chamber of a modified Boyden chamber. ECs preincubated with increasing concentrations of the antibodies were placed in the upper cell culture insert, and migration of ECs through the porous membrane was determined as described in Methods. Results are the mean±SEM of 6 to 8 separate experiments with 2 chambers per experimental condition. *P<0.05 vs EC migration in the absence of antibodies.

Effects of Anti-CD9, -ß1, and -ß3 Antibodies on Tyrosine Phosphorylation During Adhesion of ECs
Adherent ECs display high levels of phosphorylated proteins that persist throughout cell growth and in resting confluent cultures.44 Thus, when tyrosine-phosphorylated proteins were analyzed during lesion repair over a period of 48 hours in the presence of sodium orthovanadate to prevent degradation by phosphatases, the phosphorylation pattern was unchanged in the presence of anti-CD9 antibodies (50 µg/mL; data not shown). Detachment of ECs led to a sharp decrease in the tyrosine phosphorylation of proteins within 30 minutes (Figure 6aDown). When ECs were seeded in FN-coated wells, the level of tyrosine phosphorylation in cells adhering for 30 minutes increased significantly. In an identical number of adherent cells, there was no difference between ECs adhering in the presence of anti-CD9, anti-ß1, or anti-ß3 antibodies added 30 minutes before seeding (Figure 6bDown). Furthermore, identical phosphorylation patterns were observed in ECs incubated with the same antibodies for 30 minutes in suspension (data not shown).



View larger version (96K):
[in this window]
[in a new window]
  		Figure 6. Tyrosine phosphorylation during adhesion of ECs: effects of anti-CD9 (ALMA.1), anti-ß1 (4B4), and anti-ß3 (SDF.3) antibodies, RGDS peptide, and EDTA. Samples were prepared as described in Methods. a, Confluent ECs (lane 1) or ECs in suspension for 30 minutes at 37°C (lane 2). b, ECs adhering to FN after preincubation for 30 minutes at 37°C with 1% HSA (lane 1) or 50 µg/mL anti-CD9 (ALMA.1, lane 2), anti-ß1 (4B4, lane 3), or anti-ß3 (SDF.3, lane 4). c, ECs adhering after 30 minutes at 37°C to adsorbed antibodies: mouse IgG (lane 1), anti-ß1 (lane 2), anti-CD9 (lane 3), or anti-ß3 (lane 4). Deposits were equivalent to 10 000 cells in panel a and to 5000 cells in panels b and c. Photomicrographs are representative of at least 3 Western blots. Molecular weights are indicated on the right, and F indicates the migration front.

The ability of these antibodies to capture ECs was tested after adsorption of the antibodies and of control IgG on tissue-culture–grade polystyrene. ECs did not adhere to all these substrates with the same efficiency. Adhesion was significantly diminished on mouse IgG, GAM, and HSA (IgG>GAM>HSA) but was equivalent on anti-CD9 (ALMA.1), anti-ß1 (4B4), anti-ß3 (SDF.3), FN, and VN (data not shown). However, equal numbers of ECs adhering to immobilized anti-CD9, anti-ß1, or anti-ß3 antibodies or to control IgG showed similar patterns of tyrosine phosphorylation (Figure 6cUp).

CD9 Colocalizes With ß1 and ß3 in ECs
Double-labeling immunofluorescence of ECs with anti-CD9 and anti-ß1 showed common labeling of structures at the cell surface and some intracellular structures (Figure 7Down), suggesting the colocalization of CD9 with ß1 integrins not only at the cell surface (especially at cell-cell junctions) but also inside the cell, presumably with the ß1 integrin precursor.45 ß3 integrins similarly displayed colocalization with CD9. Identical experiments with anti-CD9 and anti-CD63 antibodies demonstrated that CD63 was essentially concentrated within intracellular granular structures clearly distinct from the mainly surface membrane locations of CD9.



View larger version (104K):
[in this window]
[in a new window]
  		Figure 7. Localization of CD9, CD63, ß1, and ß3 in ECs by immunofluorescence double labeling. ECs were incubated with (1) anti-CD9 (mAb ALMA.1), which was revealed by Cy3 labeling, and then anti-ß1 (mAb 4B4), which was revealed by Cy2 labeling; (2) anti-CD9, which was revealed by Cy2 labeling, and then anti-ß3 (mAb SDF.3), which was revealed by Cy3 labeling; or (3) anti-CD9, which was revealed by Cy2 labeling, and then anti-CD63 (mAb H5C6), which was revealed by Cy3 labeling, as described in Methods. Photomicrographs are representative of 8 separate experiments. Original magnification x400.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, we demonstrate that the tetraspanin CD9 participates in EC lesion repair in vitro, possibly by interacting with ß1 and ß3 integrins. Anti-CD9 antibodies inhibited cell migration and had additive effects in the presence of an anti-ß1 antibody. In addition, CD9 and ß1 were shown in double-immunolabeling experiments to localize to common structures at the surface of ECs. When cells were released from a confluent state, anti-CD9 antibodies inhibited only cell migration during lesion repair, whereas anti-ß1 antibodies affected both migration and proliferation. The effects of anti-CD9 were not mediated through Fc receptors because results could be reproduced by using F(ab')2 fragments. Finally, anti-CD9 antibodies inhibited EC migration toward FN and VN in transmembrane Boyden-type migration assays.

Angiogenesis, the sprouting of new capillaries from the preexisting vascular bed, occurs in many physiological and pathological situations and proceeds through a sequence of events typically involving proteolytic degradation of the basement membrane, migration and proliferation of ECs, lumen formation, and reconstitution of a new basal membrane.46 47 Triggering of angiogenesis requires stimulation signals, whereas temporal regulation of activators and inhibitors adds to the complexity of the process.48 The relevance of angiogenesis in tumor processes is now well established.49 Recent research has focused on the regulation of cellular signals by extracellular matrix proteins in the context of cell adhesion and angiogenesis, and integrins have been shown to play a major role in these mechanisms.26 50 51 52 In the study of cancer, the importance of the association of tetraspanins with integrins is now emerging,27 although no involvement of these complexes in angiogenesis has yet been reported in vivo. Recently, 2 studies have reported modulation by tetraspanins (CD151 and CD81) of in vitro collagen gel invasion and capillary formation, possibly through interactions with integrins or other associated molecules.53 54 Among the nearly 20 identified members of the tetraspanin family, several are expressed in ECs, with some at high levels (CD151/PETA3,54 CD9, CD63, and CD81) (Figure 1Up) and others only at low levels (CD53, A15/TALLA, and CO-029) (data not shown).

Lesion repair and angiogenesis are processes that share a number of common events, including EC migration and proliferation. Using a previously developed model of a mechanical wound in confluent ECs that preserves the neosynthesized extracellular matrix,55 we demonstrated in the present study that an anti-CD9 mAb (ALMA.1) inhibits lesion closure. Because this effect could be reproduced with F(ab')2 fragments, it would appear to be independent of Fc receptors. This is in contrast to the platelet aggregation induced by several anti-CD9 mAbs, which is mediated by the Fc{gamma}RII receptor,16 56 57 even though platelet activation specific to immobilized F(ab')2 fragments of CD9 has also been described.57

Lesion repair results from 2 complementary processes: cell migration and proliferation. Although cell adhesion precedes migration, both processes use common adhesion receptors in the form of cytoskeletal proteins and extracellular matrices, which are differently regulated.58 Anti-CD9 mAbs affected EC migration but not proliferation during lesion repair, in view of the fact that the proliferation rate at the leading edge of the lesion remained identical to that of controls. Furthermore, when irradiated ECs in which proliferation was severely hindered were used,23 anti-CD9 mAbs still inhibited lesion closure. EC adhesion was not modified by an anti-CD9 mAb (ALMA.1) under conditions in which RGDS peptide and EDTA induced significant inhibition. Moreover, this antibody did not inhibit the proliferation of sparsely seeded ECs on FN but increased it, which would further reinforce the involvement of CD9 in EC migration during lesion repair. Additional data in support of an effect on migration were obtained from transmembrane Boyden-type assays, in which an anti-CD9 mAb inhibited EC migration toward FN and VN with a potency similar to that observed in lesion repair. Specificity to CD9 was demonstrated by the lack of effect of another tetraspanin mAb, anti-CD63. However, all anti-CD9 mAbs tested did not display the same activity. Among our mAbs to CD9 (ALMA.1, ALMA.3, and RPM.13), ALMA.1 was the most potent, ALMA.3 inhibited EC migration during lesion repair to the same extent as did ALMA.1 but was inefficient in transmembrane assays, and RPM.13 did not inhibit lesion closure. These differences in activity are difficult to explain because competition experiments indicate the epitopes of these antibodies to be similar if not identical (data not shown). Nevertheless, the same antibodies also display variable agonist efficiency in platelet aggregation with a similar order of potency (ALMA.1>=ALMA.3>RPM.13) (data not shown).

The intracellular domains of tetraspanins and, hence, of CD9 would appear to be too short to induce signal transduction on their own and do not contain domains known to participate in signal transduction. Consequently, many functions assigned to these molecules must result from their association with other molecules present at the cell surface and especially with integrins.1 Integrins play a fundamental role in angiogenesis26 through their ability to interact with the extracellular matrix proteins on which ECs adhere and migrate.51 52 These interactions of integrins with the extracellular matrix mediate coupling to several signaling pathways and the recruitment of signaling molecules, such as focal adhesion kinase (FAK), Ras, and Rho.59 In the present study, we demonstrate the inhibitory effects of anti-ß1 and anti-ß3 mAbs on lesion repair and confirm the relevance of integrins to this process. Although anti-ß1 mAbs slightly ({approx}20%) inhibited the adhesion of sparsely seeded ECs under sensitive adhesion conditions, this property of ß1 would seem insufficient to account for its inhibitory effect observed on wound closure because lesion repair occurred in postconfluent adhering EC cultures. Our data further suggest that CD9, but not CD63, could cooperate with ß1 integrins in mediating EC migration for the following reasons: (1) low concentrations of anti-CD9 and anti-ß1 mAbs had additive effects on lesion repair, (2) both antibodies inhibited transmembrane migration toward FN, and (3) ß1 and CD9 colocalized to common structures on the surface of ECs. The common localization of CD9 and ß1 integrins at the cell junctions of human umbilical vein ECs was recently reported and, likewise, their coimmunoprecipitation.54 However, whereas the anti-CD9 mAb (ALMA.1) inhibited EC migration toward both FN and VN, the anti-ß1 mAb inhibited only EC migration toward FN. This indicates that CD9 could associate at least with the ß1 integrin receptors for FN and is in agreement with other reports showing that the adhesive properties of CD9 involve interactions with this protein.9 60 61 Finally, ALMA.1 also inhibited EC migration toward von Willebrand factor (data not shown). Because the VN and von Willebrand factor receptors belong to the ß3 integrins, these results suggest an association of CD9 with ß3 integrins in ECs that is similar to that occurring in platelets17 18 19 ; this was confirmed in double-immunolabeling assays in which CD9 and ß3 colocalized to some common structures of ECs. Immunoprecipitation experiments that used various cellular models like platelets, leukemic cells, keratinocytes, and Schwann cells have shown a physical proximity between CD9 and the ß1 and ß3 integrins. CD9 associates with very late antigen (VLA)-3, VLA-4, VLA-5, and VLA-6, among which VLA-3 and VLA-5 bind to FN,9 11 14 17 54 but the mechanism and type of interaction remain unknown. Our results support the most recent model of a web or lattice at the cell surface, formed by tetraspanins associated with integrins, where these tetraspanins would act as "adaptor proteins" or "molecular facilitators."1 CD9 could modulate the affinity of integrins for their ligands by direct interaction or through a yet-unidentified common stimulatory pathway.

To date, neither the type nor the mechanism of signal transduction through CD9 is known, no natural ligand has yet been described, and antibodies are the only tools available. Depending on the cell system, mAbs to CD9 appear to facilitate5 or inhibit (Reference 1111 and the present study) cell motility. Similarly, (over)expression of CD9 suppressed the motility of tumor cell lines,10 whereas its transfection into CD9-negative human B-cell lines enhanced their migratory properties.9 The effects observed in the presence of mAbs could be due to phosphorylation or dephosphorylation processes. In platelets, anti-CD9 mAbs induce an increase in the tyrosine phosphorylation of several proteins, some of which belong to the Src family and others of which have been identified as p72syk and p125FAK.15 62 Expression of CD9 in a human B-cell line increases the tyrosine phosphorylation of 69- and 130-kDa bands in cells adhering to FN,9 whereas in Schwann cells, anti-CD9 mAbs enhance the tyrosine phosphorylation of 8- to 220-kDa proteins.5

In the experimental conditions under which we observed inhibition of lesion repair, ALMA.1 did not modify the tyrosine phosphorylation profile of ECs over a period of 48 hours, even in the presence of a phosphatase inhibitor, to reveal low phosphorylation levels, nor did clustering of CD9 modify this profile (data not shown). Tyrosine-phosphorylated proteins were strongly downregulated in ECs after trypsinization and resuspension. This confirms the findings of Defilippi et al,44 who demonstrated a rapid downregulation of tyrosine-phosphorylated proteins in ECs detached from their substratum. Tyrosine phosphorylation was then increased to higher levels within 30 minutes under limiting adhesion conditions on FN and VN, and these levels were not affected by anti-CD9, anti-ß1, or anti-ß3 antibodies. In addition, identical phosphorylation patterns were observed in ECs adhering to FN, VN, or immobilized anti-CD9 or anti-integrin antibodies. These results demonstrate the following: (1) The adhesion of ECs induces high levels of protein phosphorylation (which persist in increasing) and stationary confluent cultures.44 (2) Immobilization of the receptor through matrix proteins like VN, FN, collagen I, collagen IV, and laminin44 or through other substrates, such as antibodies that allow the adhesion of ECs, induces a similar phosphorylation response. Furthermore, identical responses were obtained when these substrate molecules bound to ECs in suspension (data not shown). This is in agreement with the observation that integrin clustering, per se, in nonadherent ECs triggers p125FAK tyrosine phosphorylation.44 (3) The ability of ECs to adhere to immobilized anti-CD9 mAbs and thus promote tyrosine phosphorylation comparable to that induced by interactions with ß1 or ß3 integrins (Reference 4444 and the present study) demonstrates that CD9 participates actively in EC adhesion, either by itself or through interactions with integrins or other as-yet-unidentified proteins. As in the case of cell migration, these observations reinforce the hypothesis of the existence of cooperative mechanisms between CD9 and integrins in ECs. However, the signal(s) induced by the anti-CD9 mAb ALMA.1 during EC lesion repair still remains unknown.

In conclusion, our results point to the important role of CD9 in EC migration during lesion repair and possibly during angiogenesis. CD9 is thought to cooperate with integrins to modulate EC migration on FN and VN, and in view of the major role of ECs in angiogenesis and the relevance of this process to cancer, CD9 could represent a potential target to inhibit angiogenesis during metastatic processes.


*    Acknowledgments
 
The authors are grateful to Marlène Ehret and Sylvie Moog for excellent technical assistance, to Catherine Ravanat and Pierre Mangin for characterizing the anti-CD9 mAbs, and to Marie-Jeanne Baas for valuable advice in double-labeling immunofluorescence experiments and photographic work. We would also like to thank Dr Arnaud Charpentier of the Service de Chirurgie Cardiovasculaire des Hôpitaux Universitaires de Strasbourg for providing human saphenous vein and mammary artery fragments and Juliette Mulvihill for reviewing the English of the manuscript.

Received May 11, 1999; accepted July 12, 1999.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Maecker HT, Todd SC, Levy S. The tetraspanin superfamily: molecular facilitators. FASEB J. 1997;11:428–442.[Abstract]
  2. Wright MD, Tomlinson MG. The ins and outs of the transmembrane 4 superfamily. Immunol Today. 1994;15:588–594.[Medline] [Order article via Infotrieve]
  3. Si Z, Hersey P. Expression of the neuroglandular antigen and analogues in melanoma: CD9 expression appears inversely related to metastatic potential of melanoma. Int J Cancer. 1993;54:37–43.[Medline] [Order article via Infotrieve]
  4. Miyake M, Nakano K, Itoi S-I, Koh T, Taki T. Motility-related protein-1 (MRP-1/CD9) reduction as a factor of poor prognosis in breast cancer. Cancer Res. 1996;56:1244–1249.[Abstract/Free Full Text]
  5. Anton ES, Hadjiargyrou M, Patterson PH, Matthew WD. CD9 plays a role in Schwann cell migration in vitro. J Neurosci.. 1995;15:584–595.[Abstract]
  6. Zola H, Furness V, Barclay S, Zowtyj H, Smith M, Melo JV, Neoh SH, Bradley J. The p24 leucocyte membrane antigen: modulation associated with lymphocyte activation and differentiation. Immunol Cell Biol. 1989;67:63–70.
  7. Scherberich A, Moog S, Haan-Archipoff G, Azorsa DO, Lanza F, Beretz A. Tetraspanin CD9 is associated with very late-acting integrins in human vascular smooth muscle cells and modulates collagen matrix reorganization. Arterioscler Thromb Vasc Biol. 1998;18:1691–1697.[Abstract/Free Full Text]
  8. Lanza F, Wolf D, Fox CF, Kieffer N, Seyer JM, Fried VA, Coughlin SR, Phillips DR, Jennings LK. cDNA cloning and expression of platelet p24/CD9: evidence for a new family of multiple membrane-spanning proteins. J Biol Chem. 1991;266:10638–10645.[Abstract/Free Full Text]
  9. Shaw ARE, Domanska A, Mak A, Gilchrist A, Dobler K, Visser L, Poppema S, Fliegel L, Letarte M, Willett BJ. Ectopic expression of human and feline CD9 in a human B cell line confers ß1 integrin-dependent motility on fibronectin and laminin substrates and enhanced tyrosine phosphorylation. J Biol Chem. 1995;270:24092–24099.[Abstract/Free Full Text]
  10. Ikeyama S, Koyama M, Yamaoko M, Sasada R, Miyake M. Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA. J Exp Med. 1993;177:1231–1237.[Abstract/Free Full Text]
  11. Rubinstein E, Le Naour F, Billard M, Prenant M, Boucheix C. CD9 antigen is an accessory subunit of the VLA integrin complexes. Eur J Immunol. 1994;24:3005–3013.[Medline] [Order article via Infotrieve]
  12. Forsyth KD. Anti-CD9 antibodies augment neutrophil adherence to endothelium. Immunology. 1991;72:292–296.[Medline] [Order article via Infotrieve]
  13. Masselis-Smith A, Shaw RE. CD9-Regulated adhesion: anti-CD9 monoclonal antibody induce pre-B cell adhesion to bone marrow fibroblasts through de novo recognition of fibronectin. J Immunol. 1994;152:2768–2777.[Abstract]
  14. Hadjiargyrou M, Patterson PH. An anti-CD9 monoclonal antibody promotes adhesion and induces proliferation of Schwann cells in vitro. J Neurosci.. 1995;15:574–583.[Abstract]
  15. Ozaki Y, Satoh K, Kuroda K, Qi R, Yatomi Y, Yanagi S, Sada K, Yamamura H, Yanabu M, Nomura S, Kume S. Anti-CD9 monoclonal antibody activates p72syk in human platelets. J Biol Chem. 1995;270:15119–15124.[Abstract/Free Full Text]
  16. Kuroda K, Ozaki Y, Qi R, Asazuma N, Yatomi Y, Satoh K, Nomura S, Suzuki M, Kume S. Fc{gamma}II receptor-mediated platelet activation induced by anti-CD9 monoclonal antibody opens Ca2+ channels which are distinct from those associated with Ca2+ store depletion. J Immunol. 1995;155:4427–4436.[Abstract]
  17. Slupsky JR, Seehafer JG, Tang SC, Masellis-Smith A, Shaw AR. Evidence that monoclonal antibodies against CD9 antigen induce specific association between CD9 and the platelet glycoprotein IIb-IIIa complex. J Biol Chem. 1989;264:12289–12293.[Abstract/Free Full Text]
  18. Brisson C, Azorsa DO, Jennings LK, Moog S, Cazenave J-P, Lanza F. Co-localization of CD9 and GPIIb-IIIa ({alpha}IIbß3 integrin) on activated platelet pseudopods and {alpha}-granule membranes. Histochem J. 1997;29:153–165.[Medline] [Order article via Infotrieve]
  19. Indig FE, Diaz-Gonzalez F, Ginsberg MH. Analysis of the tetraspanin CD9-integrin {alpha}IIbß3 (GPIIb-IIIa) complex in platelet membranes and transfected cells. Biochem J. 1997;327:291–298.
  20. Nieuwenhuis HK, van Oosterhout JJG, Rozemuller E, van Iwaarden F, Sixma JJ. Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation. Blood. 1987;70:838–845.[Abstract/Free Full Text]
  21. Vischer UM, Wagner DD. CD63 is a component of Weibel-Palade bodies of human endothelial cells. Blood. 1993;82:1184–1191.[Abstract/Free Full Text]
  22. Berditchevski F, Bazzoni G, Hemler ME. Specific association of CD63 with the VLA-3 and VLA-6 integrins. J Biol Chem. 1995;270:17784–17790.[Abstract/Free Full Text]
  23. Klein-Soyer C, Beretz A, Cazenave J-P, Driot F, Maffrand J-P. Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and fibroblast growth factor. Biol Cell. 1990;68:231–238.[Medline] [Order article via Infotrieve]
  24. Klein-Soyer C, Archipoff G, Beretz A, Cazenave J-P. Opposing effects of heparin with TGF-ß or aFGF during repair of a mechanical wound of human endothelium. Influence of cAMP on cell migration. Biol Cell. 1992;75:155–162.[Medline] [Order article via Infotrieve]
  25. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27–31.[Medline] [Order article via Infotrieve]
  26. Brooks PC. Role of integrins in angiogenesis. Eur J Cancer. 1996;32A:2423–2429.
  27. Hemler ME, Mannion BA, Berditchevski F. Association of TM4SF proteins with integrins: relevance to cancer. Biochim Biophys Acta. 1996;1287:67–71.[Medline] [Order article via Infotrieve]
  28. Ruoslahti E, Haymann EG, Pierschbacher M, Engvall E. Fibronectin: purification, immunochemical properties and biological activities. Methods Enzymol. 1982;82:803–831.
  29. Preissner KT, Wassmuth R, Müller-Berghaus G. Physicochemical characterization of human S-protein and its function in the blood coagulation system. Biochem J. 1985;231:349–355.[Medline] [Order article via Infotrieve]
  30. Azorsa DO, Cazenave J-P, Lanza F. Analysis of antibodies recognizing CD9 and use of glutathione S-transferase fusion proteins to screen for monoclonal antibodies to CD9 and other members of the transmembrane 4 superfamily. In: Kishimoto T, ed. Leucocyte Typing VI. New York, NY: Garland Publishing Inc; 1997:631–632.
  31. Ravanat C, Morales M, Azorsa DO, Moog S, Schuhler S, Grunert P, Loew D, van Dorsselaer A, Cazenave J-P, Lanza F. Gene cloning of rat and mouse platelet glycoprotein V: identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage. Blood. 1997;89:3253–3262.[Abstract/Free Full Text]
  32. Hildreth JEK, Derr D, Azorsa DO. Characterization of a novel self-associating Mr 40,000 platelet glycoprotein. Blood. 1991;77:121–132.[Abstract/Free Full Text]
  33. Azorsa DO, Hyman JA, Hildreth JEK. CD63/Pltgp40: a platelet activation antigen identical to the stage specific melanoma-associated antigen ME491 Blood.. 1991;78:280–284.[Abstract/Free Full Text]
  34. Rosenfeld SI, Looney RJ, Leddy JP, Phipps DC, Abraham GN, Anderson CL. Human platelet Fc receptor for immunoglobulin G: identification as a 40,000-molecular weight membrane protein shared by monocytes. J Clin Invest. 1985;76:2317–2322.
  35. Klein-Soyer C, Cazenave J-P. Culture de cellules endothéliales vasculaires humaines. In: Sultan Y, Fischer AM, eds. Physiopathologie de l’Hémostase et de la Thrombose: Progrès en Hématologie. Paris, France: Doin Editeurs; 1986;8:83–93.
  36. Mazzucotelli J-P, Klein-Soyer C, Beretz A, Brisson C, Archipoff G, Cazenave J-P. Endothelial cell seeding: coating of Dacron and expanded polytetrafluoroethylene vascular grafts with a biological glue allows adhesion and growth of human saphenous vein endothelial cells. Int J Artif Organs. 1991;14:482–490.[Medline] [Order article via Infotrieve]
  37. Gillies RJ, Didier N, Denton M. Determination of cell number in monolayer cultures. Anal Biochem. 1986;159:109–113.[Medline] [Order article via Infotrieve]
  38. Klein-Soyer C, Beretz A, Cazenave J-P, Wittendorp-Rechenmann E, Vonesch J-L, Rechenmann RV, Driot F, Maffrand J-P. Sulfated polysaccharides modulate effects of acidic and basic fibroblast growth factors on repair of injured confluent human vascular endothelium. Arteriosclerosis. 1989;9:147–153.[Abstract/Free Full Text]
  39. Klein-Soyer C, Cazenave J-P, Herbert J-M, Maffrand J-P. SR 25989 inhibits healing of a mechanical wound of confluent human saphenous vein endothelial cells which is modulated by standard heparin and growth factors. J Cell Physiol. 1994;160:316–322.[Medline] [Order article via Infotrieve]
  40. Labourdette G, Janet T, Laeng P, Perraud F, Lawrence D, Pettmann B. Transforming growth factor-ß modulates the effects of basic fibroblast growth factor on growth and phenotypic expression of rat astroblast in vitro. J Cell Physiol. 1990;144:473–484.[Medline] [Order article via Infotrieve]
  41. Leavesley DI, Ferguson GD, Wayner EA, Cheresh DA. Requirement of the integrin ß3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen. J Cell Biol. 1992;117:1101–1107.[Abstract/Free Full Text]
  42. Leavesley DI, Schwartz MA, Rosenfeld M, Cheresh DA. Integrin ß1- and ß3-mediated endothelial cell migration is triggered through distinct signaling mechanisms. J Cell Biol. 1993;121:163–170.[Abstract/Free Full Text]
  43. Yin Z, Leprini A, Perris R, Colombantti A. Differential cation regulation of the {alpha}5ß1 integrin-mediated adhesion of leukemic cells to the central cell-binding domain of fibronectin. Cell Growth Differ. 1997;8:1339–1347.[Abstract]
  44. Defilippi P, Bozzo C, Volpe G, Romano G, Venturino M, Silengo L, Tarone G. Integrin-mediated signal transduction in human endothelial cells: analysis of tyrosine phosphorylation events. Cell Adhes Commun.. 1994;2:75–86.[Medline] [Order article via Infotrieve]
  45. Rubinstein E, Poindessous-Jazat V, Le Naour F, Billard M, Boucheix C. CD9, but not other tetraspans, associate with the ß1 integrin precursor. Eur J Immunol. 1997;27:1919–1927.[Medline] [Order article via Infotrieve]
  46. Moses MA, Langer R. Inhibitors of angiogenesis. Biotechnology. 1991;9:630–634.[Medline] [Order article via Infotrieve]
  47. Risau W. Differentiation of endothelium. FASEB J. 1995;9:926–933.[Abstract]
  48. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86:353–364.[Medline] [Order article via Infotrieve]
  49. Folkman J. What is the evidence that tumors are angiogenesis dependent ? J Natl Cancer Inst. 1990;82:4–6.[Free Full Text]
  50. Brooks PC, Clark RAF, Cheresh DA. Requirement of vascular integrin {alpha}vß3 for angiogenesis. Science. 1994;264:569–571.[Abstract/Free Full Text]
  51. Bischoff J. Approaches to studying cell adhesion molecules in angiogenesis. Trends Cell Biol. 1995;5:60–65.[Medline] [Order article via Infotrieve]
  52. Strömblad S, Cheresh DA. Cell adhesion and angiogenesis. Trends Cell Biol. 1996;6:462–468.[Medline] [Order article via Infotrieve]
  53. Sincock PM, Fitter S, Parton RG, Berndt MC, Gamble JR, Ashman LK. PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function. J Cell Sci. 1999;833–844.
  54. Yanez-Mo M, Aranzazu A, Cabanas C, Mrazuela M, Tejedo R, Ursa MA, Ashman LK, de Landazuri MO, Sanchez-Madrid F. Regulation of endothelial cell motility complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with {alpha}3ß1 integrin localized at endothelial lateral junctions. J Cell Biol. 1998;141:791–804.[Abstract/Free Full Text]
  55. Klein-Soyer C, Beretz A, Millon-Collard R, Abecassis J, Cazenave J-P. A simple in vitro model of mechanical injury of confluent cultured endothelial cells to study quantitatively the repair process. Thromb Haemost. 1986;56:232–235.[Medline] [Order article via Infotrieve]
  56. Worthington RE, Carroll RC, Boucheix C. Platelet activation by CD9 monoclonal antibodies is mediated by the Fc{gamma}II receptor. Br J Haematol. 1990;74:216–222.[Medline] [Order article via Infotrieve]
  57. Griffith L, Slupsky J, Seehafer J, Boshkov L, Shaw ARE. Platelet activation by immobilized monoclonal antibody: evidence for a CD9 proximal signal. Blood. 1991;78:1753–1759.[Abstract/Free Full Text]
  58. Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell. 1996;84:359–369.[Medline] [Order article via Infotrieve]
  59. Giancotti G. Integrin signaling: specificity and control of cell survival and cell cycle progression. Curr Opin Cell Biol. 1997;5:691–700.
  60. Jennings LK, Wilkinson DA, Crossno JT Jr. CD9 is a cell adhesion molecule on human platelets. Blood. 1995;86:282a.
  61. Crossno JT Jr, Poppleton HM, White MM, Wilkinson DA, Patterson KS, Raghow R, Jennings LK. The large extracellular loop of p24/CD9 contributes to cell motility and spreading on fibronectin. Blood.. 1995;86:282a. Abstract.
  62. Slupsky JR, Cawley JC, Kaplan C, Zuzel M. Analysis of CD9, CD32 and p67 signalling: use of degranulated platelets indicates direct involvement of CD9 and p67 in integrin activation. Br J Haematol. 1997;96:275–286.[Medline] [Order article via Infotrieve]



This article has been cited by other articles: