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10.1245/s10434-006-9009-9
Annals of Surgical Oncology 13:1241-1251 (2006)
© 2006 Society of Surgical Oncology
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Original Article

An Endogenous Inhibitor of Angiogenesis derived from a Transitional Cell Carcinoma: Clipped ß2-Glycoprotein-I

Wolf-Dietrich C. Beecken1, Tobias Engl1, Eva M. Ringel1, Kevin Camphausen2, Martin Michaelis3, Dietger Jonas1, Judah Folkman4, Yuen Shing4 and Roman A. Blaheta1

1 Department of Urology, J.W. Goethe University, Frankfurt am Main, Germany
2 Radiation Oncology Branch, National Cancer Institute, Bethesda, Maryland, USA
3 Institute for Hygiene, J. W. Goethe University, Frankfurt am Main, Germany
4 Vascular Biology Program, Children’s Hospital and Harvard Medical School, Boston, Massachusetts, USA

Correspondence: Address correspondence and reprint requests to: Roman A. Blaheta; J. W. Goethe-Universitä tsklinik, Zentrum der Chirurgie, Klinik fü r Urologie und Kinderurologie, Interdisziplinä res Forschungs- und Laborgebä ude, Haus 25, Raum 204, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany. E-mail: blaheta{at}em.uni-frankfurt.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Invasive cell carcinoma of the bladder often develops after complete transurethral excision of superficial transitional cell carcinoma. It has been postulated that primary tumors release angiogenesis-blocking proteins which suppress distant metastases. We have identified an endogenous protein which might be responsible for tumor dormancy.

Methods: A transitional cell carcinoma cell line was developed (UMUC-3i) which inhibits the growth of a tumor implant at a distant site in SCID mice. Conditioned media of UMUC-3i cultured cells was first pooled and then fractioned, and the capacity of individual components to block endothelial cell growth was tested. The protein fraction responsible for blocking endothelial cell growth was identified by N-terminal amino acid sequencing as well as by mass-spectrometry. The effects of the purified protein in preventing endothelial cell proliferation and tube formation in an in vitro angiogenesis assay was investigated.

Results: The plasma protein ß2-glycoprotein-I (ß2gpI) was isolated and identified from conditioned medium of UMUC-3i cultured cells. Based on the in vitro angiogenesis assay, ß2gpI strongly inhibited endothelial cell growth and tube formation, whereby the inhibitory activity corresponded to the clipped version of ß2gpI (cß2gpI). Clipping was induced by adding plasmin at a molar ratio 1:15 (plasmin:substrate). Further analysis indicated that cß2gpI effects were mediated by annexin II surface receptors expressed on endothelial cells.

Conclusions: cß2gpI may be involved in blocking angiogenic processes and bladder cancer progression. In this case, cß2gpI may be a promising tool in bladder cancer therapy.

Key Words: ß2-glycoprotein-I • Transitional cell carcinoma • Angiogenesis • Tumor dormancy

Abbreviations: ß2gpI: ß2glycoprotein-I • cß2gpI: clipped version of ß2gpI • TCC: Transitional cell carcinoma


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transitional cell carcinoma (TCC) of the bladder is the fourth most common malignancy that occurs in men, with by far the majority (80%) being initially superficial and easily treated by transurethral resection of the bladder tumor. Paradoxically, despite an initial complete resection of the tumor, recurrence develops in 30–90% of all cases.1,2 The progression to muscle invasive and/or metastatic stages occurs in up to 50% of the high-risk cases within 5 years of the initial diagnosis, requiring at such time radical and/or systemic therapies.

During the last decade, Folkman and coworkers demonstrated that tumor progression and metastasis are highly dependent on angiogenesis, i.e., the growth of capillary blood vessels into the tumor area.3 Based on these experiments, the term "tumor dormancy"was created to characterize the synthesis and release of anti-angiogenic proteins by the primary tumor,4,5 which keep distant metastases quiet.6 Consequently, the resection of certain primary tumors may induce rapid metastatic progression due to the loss of angioinhibitory activity.6 At the present time there is a major resurgent interest in identifying such endogenous angiogenesis inhibitors because they are relatively less toxic than conventional chemotherapy, have a lower risk of drug resistance, and, therefore, may represent a new class of specific anti-cancer agents.7,8

The objective of the investigation reported here was to obtain evidence that the plasma protein ß2-glycoprotein-I (ß2gpI; syn. apolipoprotein H) may be responsible for the dormancy of bladder carcinoma. ß2gpI was derived from conditioned media of a human transitional cell carcinoma cell line and identified by N-terminal amino acid sequencing as well as by mass-spectrometry. Based on an in vitro angiogenesis assay, ß2gpI strongly inhibited endothelial cell growth and tube formation, whereby the inhibitory activity corresponded to the clipped version of ß2gpI (cß2gpI). Further analysis indicated that cß2gpI effects were mediated by annexin II surface receptors expressed on endothelial cells.

ß2gpI is an abundant plasma protein which was first isolated in 1961.9,10 The protein has 326 amino acids and consists of four homologous repeated units of 60 amino acids (sushi domains) and a fifth domain that contains an extra 20 amino acid extension.11 The C-terminal extension in the fifth domain is surface-exposed and thereby susceptible to proteolytic cleavage, and is generated by plasmin and, to a lesser extent, by activated factor X.11,12 Several in vivo studies have demonstrated enhanced cleavage of native ß2gpI during increased fibrinolysis.13,14,15

Abnormally elevated plasma levels of ß2gpI have been linked to the activation of lipoprotein lipase,16 pro- and anticoagulant properties,17,18,19 and activation of apoptosis.20 Furthermore, ß2gpI has been identified as the primary target antigen recognized by autoantibodies in patients with the antiphospholipid syndrome (APS).21,22

Our data indicates that cß2gpI is a potential endogenous anti-cancer compound, opening the possibility of its becoming a novel tool to treat bladder cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human Umbilical Vein Endothelial Cells
Endothelial cells (HUVEC) were isolated from human umbilical veins and harvested by enzymatic treatment with chymotrypsin. HUVEC were grown in Medium 199 (M199; Biozol, Munich, Germany) supplemented with 10% fetal calf serum (FCS, Gibco, Karlsruhe, Germany), 10% pooled human serum, 20 µg/ml endothelial cell growth factor (Boehringer, Mannheim, Germany), 0.1% heparin, 100 ng/ml gentamaycin, and 20 mM HEPES-buffer (pH 7.4). To control the purity of HUVEC cultures, cells were stained with fluorescein isothiocyanate (FITC)-labeled monoclonal antibody against Factor VIII-associated antigen (Von Willebrand factor; clone F8/86; Dako, Hamburg, Germany) and analyzed microscopically or by FACscan (Becton Dickinson, Heidelberg, Germany; FL-1H (log) channel histogram analysis; 1 x 104 cells/scan). Cell cultures with a purity >95% were serially passaged. Subcultures from passages 2–4 were selected for experimental use.

Human foreskin fibroblasts (HFF) served as the control culture system. HFF were grown in DMEM/HAM’s F12 medium (Biochrom AG, Berlin) supplemented with 10% FCS, 2% Glutamax, 1% penicillin/streptomycin, and 20 mM HEPES-buffer (pH 7.4).

Selection of the UMUC-3 Human Transitional Cell Carcinoma Cell Line
By selective in vivo passage, we were able to develop a variant (UMUC-3i) of the UMUC-3 human transitional cell carcinoma cell line in which a primary flank tumor almost completely suppressed the growth of a second implant on the opposite flank, as has previously been shown.5

Collection of Conditioned Media
The UMUC-3i cell line was established in vitro and grown in 900-cm2 roller bottles (Corning Costa, Corning, N.Y.) containing 80 ml of Dulbecco’s modified Eagle’s medium (DMEM; JRH Bioscience, Lenexa, Canada) with 3% heat inactivated fetal calf serum (Intergene, Purchase, N.Y.) and 1% glutamine penicillin-streptomycin (Irvine Scientific, Santa Ana, Calif.). After 72 h at 37°C and 10% CO2, the media were collected, centrifuged, filtered (0.22 µm; Corning Costar, Rochester, N.Y.), and stored at 4°C.

Protein Purification
Pooled conditioned media were diluted 1:3 with sterile 10 mM tris-HCl pH 7.4 and applied to an 80-ml heparin Sepharose column (Pharmacia, Uppsala, Sweden). After washing with 10 mM Tris-HCl, pH 7.4, the column was eluted with a continuous gradient from 50 mmol to 1.0 M NaCl in 10 mM Tris-HCl, pH 7.4, and 2-ml fractions were collected. The inhibitory activity of all fractions was tested by the capillary endothelial cell proliferation assay. Active fractions were pooled and applied to a SynChropak C4-reversed-phase high-performance liquid chromatography (HPLC) column (C4-RP column; Eichrom Industries., Darien, Ill.) equilibrated with H2O and 0.1% trifluoroacetic acid (TFA). After washing with H2O and 0.1% TFA, protein was eluted with a continuous gradient of acetonitrile in TFA, and fractions were collected again. The fractions were then evaporated by vacuum centrifugation, resuspended in 200 µl phosphate buffered saline (PBS), and applied to the capillary endothelial cell proliferation assay. Active fractions from several batches were pooled and reapplied to the C4-RP column. Using a shallower gradient of acetonitrile, the protein was eluted again and evaporated, and the resuspended fractions were tested on the capillary endothelial cell proliferation assay. After three cycles of C4-RP HPLC, the inhibitory activity was purified to homogeneity.

Capillary Endothelial Cell Proliferation Assay
Bovine capillary endothelial cells were trypsinized and suspended in DMEM containing 10% bovine calf serum (BCS; GibcoBRL, Grand Island, N.Y.) and 1% glutamine penicillin-streptomycin. The cell number was adjusted to 25,000 per milliliter, and 0.5 ml of the cell suspension was pipetted into each well of a 24-well plate (Costa Corning). The cells were then allowed to settle and grow for 24 h. The media were changed to DMEM containing 5% BCS, 1% glutamine penicillin-streptomycin, and 1 ng/ml of basic fibroblast growth factor (bFGF; Scios Nova, Mountain View, Calif). Aliquots of 10 µl of each fraction from the protein purification were added to the assay. After a 72-h incubation, the cells were trypsinized, resuspended in Hematall buffer solution (Fisher Scientific) and counted with a Coulter particle counter (Coulter, Miami, Fla.). The results were compared to standard controls.

N-terminal Amino Acid Sequencing of the Inhibitory Protein
N-terminal sequencing was performed by automated Edman degradation on a Procise 494cLC protein sequencer (Perkin Elmer-Applied Biosystems Division, Foster City, Calif.) with high-sensitivity phenylthiohydantoin amino acid detection by capillary HPLC. A sequence library search and comparison were performed against combined GenBank, Brookhaven Protein, and SWISS-PROT databases.

Mass-Spectrometry of Trypsin Digest fragments of the Inhibitory Protein
The trypsin digest fragments of the inhibitory protein were identified by mass-spectrometry as described elsewere.23 The protein was identified based on fragment sizes, which were entered into the Mascot database. The probability score for the nine identified fragments originating from ß2gpI was 187 (a score of 67 indicates a probability of P < 0.05).

Plasmin Digest of Human ß2-Glycoprotein-I
Human ß2gpI was derived from CellSystems Biotechnology (St. Katharinen, Germany). A variation of the method of Okhura et al.13 and Guerin et al.24 was used in which ß2gpI (50 µg) was incubated with 5.3 µg plasmin, with a molar ratio of plasmin to substrate of 1:15. The reaction took place in serum-deprived HUVEC medium at 37°C for 2 h and was terminated by snap freezing the digests in dry ice. In all experiments, the effects of clipped ß2gpI were compared with the effects of non-clipped material, i.e., (1) untreated ß2gpI; (2) plasmin; (3) control medium.

Annexin II Monoclonal Antibody
An anti-annexin II monoclonal antibody was used for the blocking studies (clone 5; Becton Dickinson, Franklin Lakes, N.J.). Prior to use, 200 µl of antibody solution was dialyzed for 5 h using a Slide-A-Lyzer cassette (Perbio Science, Bonn, Germany) in order to remove NaN3 and to prevent toxic effects which may have been evoked by this chemical adjuvant. The antibody was finally diluted 1:5 or 1:10 in HUVEC medium.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblot
Aliquots were subjected to SDS-PAGE under non-reducing conditions on a 10% gradient gel, and proteins were subsequently visualized by Coomassie Blue staining of the gels. For Western blot analysis, protein lysates were applied to a 10 % polyacrylamide gel and electrophoresed for 90 min at 80 V. The protein was then transferred to nitrocellulose membranes. After blocking, the membranes were incubated overnight with a ß2gpI antibody (goat anti-human, CL20021AP; Biozol Diagnostika, Eching, Germany; dilution 1:100). HRP-conjugated rabbit-anti-goat IgG (Upstate Biotechnology, Lake Placid, N.Y.; dilution 1:5000) served as the secondary antibody. The membrane was briefly incubated with ECL detection reagent (ECL; Amersham, Buckinghamshire, UK) to visualize the proteins and then exposed to an X-ray-film (Hyperfilm EC; Amersham).

Human Umbilical Vein Endothelial Cell (HUVEC) Proliferation Assay
The HUVEC were adapted to HUVEC medium containing 2% fetal bovine serum (Endothelial Cell Basal Medium; Cambrex, Apen, Germany) and then seeded onto 6-well plates (0.5 x 106 cells/ml) in the presence of test materials containing (1) untreated ß2gpI, (2) ß2gpI treated with plasmin, (3) plasmin, and (4) culture medium without supplements (control). After a 24-h incubation, the cells were collected, stained with 0.4% trypan blue, and counted using a hemocytometer. In parallel experiments, cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). HUVEC media (100 µl, 5 x 104 cells/ml) were seeded onto 96-well tissue culture plates and incubated as described above. After 24 h, MTT (0.5 mg/ml) was added and the media incubated for an additional 4 h. Thereafter, cells were lysed in a buffer containing 10% SDS in 0.01 M HCl. The plates were allowed to stand overnight at 37°C, 5% CO2. Absorbance at 570 nm was determined for each well using a microplate ELISA reader. After background absorbance was subtracted, the results were expressed as cell number.

Tube Formation Assay
This assay was performed as described previously.25 Briefly, 96-well plates were coated with cold Matrigel (50 µl/well) which was allowed to polymerize at room temperature for about 30 min. Thereafter, 100 µl of a suspension of HUVEC (5 x 104 cells/ml) was seeded onto the Matrigel and cultured overnight in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 1% (v/v) fetal calf serum (FCS), and 5 ng/ml basic fibroblast growth factor (bFGF). Tube formation was assessed after 12 hours and quantified by determining the number of branching points.

Analysis of Annexin II Surface Expression
HUVEC were washed in blocking solution [PBS, 0.5% bovine serum albumin (BSA)] and then incubated for 60 min at 4°C with a mouse anti-annexin II monoclonal antibody (isotype: IgG1, clone: 5; Becton Dickinson). Fluoresceinisothiocyanate (FITC) served as the secondary antibody. Annexin II surface expression of HUVEC was then measured using a FACscan (Becton Dickinson; FL-1H (log) channel histogram analysis; 1 x 104 cells/scan) and expressed as mean fluorescence units (MFU). Mouse IgG1-FITC was used as the isotype control.

To analyze annexin II localization on the cell membrane, HUVEC cultures were transferred to round cover slips (pretreated with 2% 3-aminopropyl-triethoxysilan) placed in a 24-well multiplate. Upon reaching confluency, cell cultures were washed twice with PBS (with Ca2+ and Mg2+) and then fixed in cold (–20°C) methanol/acetone (60/40, v/v). The cells were then washed again with PBS (without Ca2+ and Mg2+), followed by one washing with blocking buffer (0.5% BSA in PBS without Ca2+ and Mg2+). After the washing buffer was removed, HUVEC cultures were incubated for 60 min with FITC-conjugated anti-annexin II monoclonal antibody. To prevent photobleaching of the fluorescent dye, cover slips with the stained cells were taken out of the wells and the residual liquid removed. The cells were then embedded in an antifade reagent/mounting medium mixture (ProLong Antifade kit; MoBiTec, Gö ttingen, Germany) and mounted on slides. The slides were then viewed using a confocal laser scanning microscope (LSM 10; Zeiss, Jena, Germany) with a planneofluar x100 /1.3 oil immersion objective.

Statistics
Each experiment was performed three to six times. Statistical significance was investigated using the Wilcoxon-Mann-Whitney U-test. Differences were considered to be statistically significant at a P value of less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Purification and Identification of ß2gpI
In pilot experiments, the efficacy of seven different transitional cell carcinoma cell lines were screened for their ability to inhibit the growth of a tumor implant grown in the flank of SCID mice. One cell line, UMUC-3, inhibited the growth of a secondary implant by 66%. By selective in vivo passage, a variant of this cell line was developed (UMUC-3i) which blocked tumor growth at the distant site by 95% (Fig. 1Go).


Figure 1
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  		FIG. 1. The bottom part of this figure illustrates five mice of the selection experiment (fourth selection passage) with the UMUC-3 transitional cell carcinoma cell line. While one tumor was growing on the mouse flank, the implant on the opposite side (arrows) was inhibited by 95%. The upper part of the figure shows the growing and blocked tumors after explanation.

 
After UMUC-3i tumor cells were re-established in vitro, conditioned media were collected and protein purification was performed. Using heparin sepharose FPLC and reversed-phased HPLC column chromatography and then applying the collected fractions to a standard capillary endothelial cell proliferation assay,8 we succeeded in identifying strong endothelial cell inhibitory activity; the active agent was purified to homogeneity.

The activity eluted between 300 and 400 mmol NaCl on heparin sepharose chromatography. After applying pooled active fractions from heparin sepharose chromatography to a C4-reversed phase HPLC column, the activity eluted at 40% acetonitrile. Within three cycles of C4-reversed phase HPLC chromatography, the inhibitory activity was localized to a single 52-kDa band by SDS-PAGE under reducing conditions (Figs. 2Go, 3Go). N-terminal amino acid sequencing as well as mass-spectrometry of the trypsin digest fragments of the protein enabled the inhibitor to be identified as ß2gpI (Fig. 3Go).


Figure 2
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  		FIG. 2. Representative endothelial cell proliferation assay showing the endothelial cell inhibitory activity of the purified protein from conditioned media of the UMUC-3i transitional cell carcinoma cell line. After heparin-Sepharose and three cycles of C4-reversed-phase chromatography the inhibitory activity was apparent only in fraction 24.

 

Figure 3
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  		FIG. 3. Representative SDS-PAGE (reduced, silver stain) of fraction 24, demonstrating a single protein band. Protein contained in fraction 24 was identified by mass-spectrometry of trypsin-digested fragments and N-terminal amino acid sequencing. Both methods consistently revealed ß2gpI as the most likely candidate protein in fraction 24. Sequence library searches in the GenBank, Brookhaven Protein, and SWISS-PROT databases of the identified N-terminal amino acids sequence (framed capital letters) did not reveal any other protein containing the identified sequence. The mass-spectrometry of trypsin-digested fragments (small letters indicate the complete aa sequence of ß2gpI, red and underlined letters indicate the identified fragments) of the protein contained in fraction 24 confirmed the result of the N-terminal amino acid sequence with a probability score of 187 (a probability score of 67 indicates a probability of P < 0.05).

 
Characterization of the Endothelial Cell Inhibitory Activity of Human ß2-lycoprotein-I
To determine whether the identified protein was indeed responsible for the endothelial cell inhibition, endothelial cell assays were conducted using commercially available human ß2gpI. The human ß2gpI did not show inhibitory activity on endothelial cell proliferation (Fig. 4Go). Possible explanations for this may be that human ß2gpI is not an endothelial cell inhibitor or that the native protein was modified to exert endothelial cell inhibitory activity.


Figure 4
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  		FIG. 4. Influence of cß2gpI on HUVEC proliferation. HUVEC growing in cell culture medium containing 2% FBS were applied to (1) culture medium alone (control), (2) untreated ß2gpI, (3) plasmin, and (4) ß2gpI treated with plasmin. In parallel experiments, annexin II receptors were blocked by pretreating HUVEC with 50 µg/ml anti-annexin II monoclonal antibody for 120 min. The ß2gpI/plasmin mixture was subsequently added. Two assays, both of which measure the cell number of HUVEC, were used to assess endothelial cell proliferation after 24 h. The cells were first collected from each well, stained with 0.4% trypan blue, and counted using a hemocytometer (Neubauer assay). As a second method, cell proliferation was assessed using the MTT dye reduction assay and photometric quantification. The Y-axis of the graph indicates cell number depicted x105 (Neubauer assay) or x104 (MTT-assay). (*P < 0.05 for ß2gpI/plasmin versus untreated HUVEC; **P < 0.05 for ß2gpI/plasmin versus HUVEC pretreated with anti annexin II; n.d. = not done).

 
We proposed in an earlier publication that cryptic protein fragments could regulate angiogenesis,4,5,26 and it has recently been reported that plasmin cleaves ß2gpI.11,24 Based on this latter finding together with our earlier proposal, we hypothesized that plasmin may generate a clipped form of ß2gpI that could exert endothelial cell inhibitory activity.

When human plasmin was incubated for 120 min with human ß2gpI at a molar ratio of plasmin to substrate of 1:15, SDS-PAGE analysis revealed an increase in ß2gpI mobility compared with that of intact ß2gpI (Fig. 5Go). The effect could not be enhanced further when plasmin was incubated with ß2gpI at a molar ratio of 1:5 (data not shown), although weaker effects were seen at a molar ratio of 1:50 (data not shown). A new, small protein band appeared in Western blot analyses of ß2gpI-plasmin mixtures (1:15 molar ratio) compared to the band of pure ß2gpI (Fig. 5Go). The small protein band did not reveal plasmin content since the pure plasmin solution did not react with the anti-ß2gpI monoclonal antibody CL20021AP. Therefore, it was concluded that the small protein band displays the clipped version of human ß2gpI (cß2gpI).


Figure 5
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  		FIG. 5. (A) SDS-PAGE analysis of ß2gpI digests. Aliquots of the specimen were taken after 2 h and subjected to SDS-PAGE analysis under non-reducing conditions on a 10% gradient gel. The resulting Coomassie Blue-stained gel is shown. Bands are related to untreated ß2gpI, to plasmin, and to the plasmin/ß2gpI reaction mixture. An increase in mobility is demonstrated for the plasmin/ß2gpI reaction mixture. MW = molecular weight marker. (B) SDS--PAGE and immunoblot of ß2gpI treated with or without plasmin. Protein lysates were applied to a 10% SDS-PAGE under non-reducing conditions. Transferred proteins were stained overnight with goat anti-human antibody, clone CL20021AP, dilution 1:100, and HRP-conjugated rabbit-anti-goat IgG antibodies.

 
Applying the reaction solution to endothelial cells strongly and significantly inhibited cell growth as revealed by two different in vitro proliferation assays (Figure 4Go). Untreated ß2gpI, as well as untreated plasmin, did not evoke proliferation blocking effects, compared to control cultures (Fig. 4Go). Furthermore, cß2gpI abolished tube formation in an endothelial cell matrigel assay (Fig. 6Go). The mean number of branching points decreased from 118 (controls; minimum: 111, maximum: 125; n = 3) to < 10 (mean: 8 ± 2%; n = 3), whereas the native proteins did not modulate HUVEC outgrowth compared to controls. To demonstrate the specificity of cß2gpI, we repeated the experiments using human foreskin fibroblasts. None of the applied mixtures influenced the proliferation of human foreskin fibroblasts.


Figure 6
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  		FIG. 6. (A) Influence of ß2gpI and plasmin on tube formation of human umbilical vein endothelial cells. 96-well plates were coated with cold Matrigel which was allowed to polymerize at room temperature for about 30 min. Subsequently, HUVEC (5 x 104 cells/ml) were seeded onto the Matrigel and cultured overnight. HUVEC were pretreated with ß2gpI, plasmin, or with plasmin + ß2gpI at a molar ratio of plasmin to substrate of 1:15. In parallel experiments, HUVEC were incubated with anti-annexin II monoclonal antibody before adding the plasmin + ß2gpI mixture. Representative photographs show the inhibition of tube formation after treatment with ß2gpI + plasmin, and reversible effects when HUVEC were preincubated with anti-annexin II monoclonal antibodies at a 1:5 dilution. Incubation with anti-annexin II monoclonal antibodies alone did not influence tube formation. (B) Quantification of the number of branching points. Controls were set to 100% (mean ± SD of n = 4 experiments).

 
The Endothelial Cell Inhibitory Effect of Clipped Human ß2-Glycoprotein-I is Mediated via Annexin II
It is known that ß2gpI binds to endothelial cells; however, the ligand of ß2gpI has not been characterized in detail and was assumed to result from the association of ß2gpI with endothelial membrane phospholipids.27,28 High-affinity binding between annexin II and ß2gpI has recently been reported, indicating that annexin II could mediate the binding of ß2gpI to endothelial cells.29,30 Flow cytometry demonstrated a significant and specific surface expression of annexin II on endothelial cells, but not on human foreskin fibroblasts (Fig. 7Go). Annexin II accumulated predominately at the cell-cell boundaries (Fig. 7Go). Following fluorescence-activated cell sorting of human foreskin fibroblasts, annexin II was not detected by confocal microscopy.


Figure 7
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  		FIG. 7. (A) Annexin II surface expression analysis. HUVEC or HFF were incubated with a FITC-conjugated mouse anti-annexin II monoclonal antibody (isotype: IgG1, clone: 5). Annexin II was measured using a FACscan [FL-1H (log) channel histogram analysis; 1 x 104 cells/scan] and expressed as mean fluorescence units (MFU). Mouse IgG1-FITC (GAM) served as the isotype control. Significant amounts of annexin II receptors were detected on HUVEC (P < 0.05), whereas no differences were measured between IgG1-FITC (GAM) and anti-annexin II treated HFF. (B) Confocal analysis of annexin II distribution. HUVEC were grown in standard medium. FITC-conjugated monoclonal antibody clone 5 was used to analyze annexin II localization. The figure shows distinct annexin II expression at the intercellular boundaries. Scale = 10 µM. x100/1.3 oil immersion objective.

 
To determine whether cß2gpI inhibition of endothelial cell proliferation and tube formation are mediated via the annexin II receptor, endothelial cells were pretreated with a specific monoclonal antibody against annexin II (CL20021AP) before cß2gpI was added. CL20021AP was found to prevent the ability of cß2gpI to inhibit endothelial cell growth, and it restored tube formation (Figs. 4Go, 6Go).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our experimental results show that human bladder cancer can convert circulating ß2gpI to a clipped version (cß2gpI) that inhibits endothelial cell proliferation and tube formation. The effect of cß2gpI on endothelial cell growth was found to be mediated by the annexin II surface receptor.

Dormancy of tumor growth and metastastic progression are well recognized clinical phenomena in cancer patients.31 O’Reilly and others have shown that the generation of angiogenesis inhibitors by a primary tumor may provide a possible mechanism for metastatic dormancy.6,32

Human TCC depends strongly on angiogenesis.7,8 While surgical removal of the tumor remains the treatment of choice, 40% of all patients with invasive TCC who are treated by radical cystectomy die within 5 years due to metastatic disease.33 Almost 50% of these patients develop rapid metastatic progression within 12–18 months after surgical excision of the primary bladder tumor.33 The clinical course of such patients is consistent with that found in animal models in which removal of a primary tumor decreases circulating levels of an endogenous angiogenesis inhibitor originally generated by the primary tumor and is followed by rapid growth of remote metastases.4,6,32,34,35

The evidence presented here shows that the primary tumors of TCC patients may actually control the malignant growth of daughter tumors. Based on our results, we conclude that primary TCC tumors synthesize the anti-angiogenic protein ß2gpI, which in turn prevents neovascularization into the secondary tumor. However, the native protein does not exert any effect on endothelial cells. Rather, the generation of the clipped version of ß2gpI by human plasmin is necessary in order to evoke a significant blockade of endothelial cell proliferation and tube formation.

Current knowledge of ß2gpI is still limited. ß2gpI has been described as an endothelial cell viability factor,36 and it is know to be produced by endothelial cells37 and to bind specifically and with high affinity to endothelial cells.38 ß2gpI seems to be the major target antigen for antiphospholipid antibodies which play a crucial role in the pathogenesis of the anti-phospholipid syndrome.39 However, no specific function has been ascribed to this particular molecule, and the physiological/pathological relevance of the chemical conversion of ß2gpI into cß2gpI is not clear.

cß2gpI has recently been reported to suppress ß2gpI-evoked thrombin activation.11 Furthermore, the binding of negatively charged surfaces (e.g., phospholipid) to ß2gpI was dramatically reduced following ß2gpI cleavage.12 Based on this, it was assumed that cleavage of ß2gpI reduces the amount of native ß2gpI, and consequently, overall ß2gpI activity.24

The concept of regulators of angiogenesis derived as cryptic fragments from the coagulation cascade and fibrinolytic system has been described for angiostatin, endostatin, antiangiogenic at-III and many more components of the system.26 ß2gpI circulates in native form in human plasma, whereby the C-terminal extension is surface-exposed and susceptible to proteolytic cleavage.11,12 In this context, plasmin seems the most feasible candidate for in vivo cleavage of ß2gpI.11,15 Most likely, the nicked form of ß2gpI is generated on the surface of cells or on the extracellular matrix–but not in the fluid phase of the blood: (1) plasminogen, the precursor of plasmin, binds to the extracellular matrix and/or fibrin to become activated;40,41 (2) the proteolytic activity of plasmin is induced on the extracellular matrix as well.40 Accordingly, cß2gpI is found in human plasma in pathological states of increased fibrinolysis.13,14,15 Disseminated intravasal coagulation and the activation of fibrinolysis is a well-known process in human cancers.41 In transitional cell carcinoma of the bladder, the detection of fibrinolytic markers is common and is used as a diagnostic tool.42

Since high-affinity binding of ß2gpI to endothelial cells has been shown to be mediated by annexin II,29 we examined whether the anti-proliferative effect of cß2gpI is coupled to this cell surface receptor. Indeed, blocking of annexin II completely abrogated the anti-endothelial effect of cß2gpI, indicating that annexin II is the specific ligand for cß2gpI. Annexins belong to a multigene family of more than 20 distinct proteins.43 Annexin II has been implicated in membrane aggregation, membrane fusion, and membrane organization during exocytotic and endocytotic processes.44,45,46 However, it also serves as a trans-membranous cell surface receptor to bind the plasminogen activator tenascin-c.47,48 Interestingly, tenascin-c has been implicated in the stimulation of angiogenesis, notably in breast cancer.49 Although purely speculative, tenascin-c could stimulate–while cß2gpI could inhibit–angiogenic processes by specific occupation of the annexin II receptor.

Ongoing studies will deal with the mode of action of cß2gpI in blocking angiogenesis. Preliminary data refer to annexin II-triggered alterations of proteins involved in cell-cycle regulation and to modulations in the MAPK pathway.

In summary, an anti-angiogenic function has been ascribed to cß2gpI which seems to be exerted by the target receptor annexin II expressed on endothelial cells. This opens up the intriguing option that external application of cß2gpI may slow the progression of transitional cell carcinoma. Ongoing studies are currently exploring the diagnostic as well as the therapeutic potential of this protein.


   ACKNOWLEDGMENTS
 
We would like to thank Karen Nelson for critically reading the manuscript.

Received for publication October 27, 2005. Accepted for publication December 13, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Stein JP, Grossfeld GD, Ginsberg DA, Esrig D, Freeman JA, Figueroa AJ, Skinner DG, Cote RJ. Prognostic markers in bladder cancer: A contemporary review of the literature. J Urol 1998; 160:645–59.[CrossRef][Medline]
  2. Oosterlinck W, Lobel B, Jakse G, Malmstrom PU, Stockle M, Sternberg C. Guidelines on bladder cancer. Eur Urol 2002; :41105–112.
  3. Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med 2003; 3:643–51.[CrossRef][Medline]
  4. O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses MA, Lane WS, Cao Y, Sage BH, Folkman J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994; 79:315–28.[CrossRef][Medline]
  5. O’Reilly MS, Pirie-Shepherd S, Lane WS, Folkman J. Anti-angiogenic activity of the cleaved conformation of the serpin antithrombin. Science 1999; 285:1926–28.[Abstract/Free Full Text]
  6. Holmgren L, O’Reilly MS, Folkman J. Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med 1995; 1:149–53.[CrossRef][Medline]
  7. Beecken W-D, Fernandez A, Joussen AM, Achilles E-G, Flynn E, Lo K-M, Gillies SD, Javaherian K, Folkman J, Shing Y. Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice. J Natl Cancer Inst 2001; 93:382–87.[Abstract/Free Full Text]
  8. Beecken W-D, Fernandez A, Panigrahy D, Achilles E-G, Kisker O, Flynn E, Joussen AM, Folkman J, Shing Y. Efficacy of antiangiogenic therapy with TNP-470 in superficial and invasive bladder cancer models in mice. Urology 2000; 56:521–26.[CrossRef][Medline]
  9. Day JR, O’Hara PJ, Grant FJ, Lofton-Day C, Berkaw MN, Werner P, Arnaud P. Molecular cloning and sequence analysis of the cDNA encoding human apolipoproteinH (ß2-Glyko-protein-1). Int J Clin Lab Res 1992; 21:256–63.[Medline]
  10. Schultze HE, Heide K, Haupt H. ü ber ein bisher unbekanntes niedermolekulares ß2-Globulin des Humanserums. Naturwissenschaften 1961; 148:719.
  11. Shi T, Iverson GM, Qi JC, Cockerill KA, Linnik MD, Konecny P, Krilis S. ß2-Glykoprotein-1 binds factor XI and inhibits its activation by thrombin and factor XIIa: Loss of inhibition by clipped ß2-glykoprotein-1. Proc Natl Acad Sci USA 2004; 101:3939–943.[Abstract/Free Full Text]
  12. Sheng Y, Sali A, Herzog H, Lahnstein J, Krilis SA. Site-directed mutagenesis of recombinant human beta 2-glycoprotein I identifies a cluster of lysine residues that are critical for phospholipid binding and anti-cardiolipin antibody activity. J Immunol 1996; 157:744–51.
  13. Ohkura N, Hagihara Y, Yoshimura T, Goto Y, Kato H. Plasmin can reduce the function of human beta2 glycoprotein I by cleaving domain V into a nicked form. Blood 1998; 91:4173–79.[Abstract/Free Full Text]
  14. Horbach DA, van Oort ET, Lisman T, Maijers JC, Derksen RH, de Groot PG. Beta2-glycoprotein I is proteolytically cleaved in vivo upon activation of fibrinolysis. Thromb Haemostasis 1999; 81:87–95.[Medline]
  15. Itoh Y, Inuzuka K, Kohno I, Wada H, Shiku H, Ohkura N, Kato H. Highly increased plasma concentrations of the nicked form of beta(2) glycoprotein I in patients with leukemia and with lupus anticoagulant: measurement with a monoclonal antibody specific for a nicked form of domain V. J Biochem (Tokyo) 2000; 128:1017–24.[Abstract/Free Full Text]
  16. Nakaya Y, Schaefer EJ, Brewer HB Jr. Activation of human post heparin lipoprotein lipase by apolipoprotein H (beta 2-glycoprotein I). Biochem Biophys Res Commun 1980; 95:1168–72.[CrossRef][Medline]
  17. Shi W, Chong BH, Hogg PJ, Chesterman CN. Anticardiolipin antibodies block the inhibition by beta 2-glycoprotein I of the factor Xa generating activity of platelets. Thromb Haemostasis 1993; 70:342–5.[Medline]
  18. Nimpf J, Bevers EM, Bomans PH, Till U, Wurm H, Kostner GM, Zwaal RF. Prothrombinase activity of human platelets is inhibited by beta 2-glycoprotein-I. Biochim Biophys Acta 1986; 884:142–149.[Medline]
  19. Nimpf J, Wurm H, Kostner GM. Beta 2-glycoprotein-I (apo-H) inhibits the release reaction of human platelets during ADP-induced aggregation. Atherosclerosis 1987; 63:109–14.[CrossRef][Medline]
  20. Manfredi AA, Rovere P, Heltai S, Galati G, Nebbia G, Tincani A, Balestrieri G, Sabbadini MG. Apoptotic cell clearance in systemic lupus erythematosus. II. Role of beta2-glycoprotein I. Arthritis Rheum 1998; 41:215–23.[CrossRef][Medline]
  21. McNeil HP, Simpson RJ, Chersterman CN, Krilis SA. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990; 87:4120–4.[Abstract/Free Full Text]
  22. Galli M, Comfurius P, Maassen C, Hemker HC, de Baets MH, van Breda-Vriesman PJ, Barbui T, Zwaal RF, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990; 335:1544–7.[CrossRef][Medline]
  23. Shevchenko A, Chernushevich I, Wilm M, Mann M. De Novo peptide sequencing by nanoelectrospray tandem mass spectrometry using triple quadrupole and quadrupole/time-of-flight instruments. Methods Mol Biol 2000; 146:1–16.[Medline]
  24. Guerin J, Sheng Y, Reddel S, Iverson GM, Chapman MG, Krilis SA. Heparin inhibits the binding of beta 2-glycoprotein I to phospholipids and promotes the plasmin-mediated inactivation of this blood protein. Elucidation of the consequences of the two biological events in patients with the anti-phospholipid syndrome. J Biol Chem 2002; 277:2644–9.[Abstract/Free Full Text]
  25. Michaelis M, Michaelis UR, Fleming I, Suhan T, Cinatl J, Blaheta RA, Hoffmann K, Kotchetkov R, Busse R, Nau H, Cinatl J Jr. Valproic acid inhibits angiogenesis in vitro and in vivo. Mol Pharmacol 2004; 65:520–7.[Abstract/Free Full Text]
  26. Browder T, Folkman J, Pirie-Shepherd S. The hemostatic system as a regulator of angiogenesis. J Biol Chem 2000; 275:1521–4.[Free Full Text]
  27. Wurm H. beta 2-Glycoprotein-I (apolipoprotein H) interactions with phospholipid vesicles. Int J Biochem 1984; 16:511–5.[CrossRef][Medline]
  28. Polz E, Kostner GM. Binding of beta 2-glycoprotein-I to in-tralipid: determination of the dissociation constant. Biochem Biophys Res Commun 1979; 90:1305–12.[Medline]
  29. Ma K, Simantow R, Zhang J-C, Silverstein R, Hajjar KA, McCrae KR. High affinity binding of ß2-glycoprotein I to human endothelial cells is mediated by annexin II. J Biol Chem 2000; 275:15541–48.[Abstract/Free Full Text]
  30. Zhang J, McCrae KR. Annexin A2 mediates endothelial cell activation by antiphospholipid/anti-beta2 glycoprotein I antibodies. Blood 2005; 105:1964–69.[Abstract/Free Full Text]
  31. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995; 1:27–31.[CrossRef][Medline]
  32. O’Reilly MS, Holmgren L, Chen C, Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med 1996; 2:689–92.[CrossRef][Medline]
  33. Rü bben H, Otto T. Lokal fortgeschrittenes oder metastasiertes Harnblasenkarzinom. Aktuelle Aspekte in der Therapie. Urologe (A) 2001; 40:464–67.
  34. Westphal JR, Van’t Hullenaar R, Geurts-Moespot A, Sweep FC, Verheijen JH, Bussemakers MM, Askaa J, Clemmensen I, Eggermont AA, Ruiter DJ, De Waal RM. Angiostatin generation by human tumor cell lines: involvement of plasminogen activators. Int J Cancer 2000; 86:760–67.[CrossRef][Medline]
  35. Campbell SC, Volpert OV, Ivanovich M, Bouck NP. Molecular mediators of angiogenesis in bladder cancer. Cancer Res 1998; 58:1298–304.[Abstract/Free Full Text]
  36. Cai G, Satoh T, Hoshi H. Purification and characterization of an endothelial cell-viability maintaining factor from fetal bovine serum. Biochim Biophys Acta 1995; 1269:13–8.[Medline]
  37. Caronti B, Calderaro C, Alessandri C, Conti F, Tinghino R, Palladini G, Valesini G. Beta2-glycoprotein I (beta2-GPI) mRNA is expressed by several cell types involved in anti-phospholipid syndrome-related tissue damage. Clin Exp Immunol 1999; 115:214–9.[CrossRef][Medline]
  38. Knudsen BS, Silverstein RL, Leung LL, Harpel PC, Nachman RL. Binding of plasminogen to extracellular matrix. J Biol Chem 1986; 261:10765–71.[Abstract/Free Full Text]
  39. Li Z, Krilis SA. Anti-beta(2)-glycoprotein I antibodies and the antiphospholipid syndrome. Autoimmun Rev 2003; 2:229–34.[CrossRef][Medline]
  40. Wu HL, Chang BI, Wu DH, Chang LC, Gong CC, Lou KL, Shi GY. Interaction of plasminogen and fibrin in plasminogen activation. J Biol Chem 1990; 265:19658–64.[Abstract/Free Full Text]
  41. Gouin-Thibault I, Achkar A, Samama MM. The thrombophilic state in cancer patients. Acta Haematol 2001; 106:33–42.[CrossRef][Medline]
  42. Tsihlias J, Grossman HB. The utility of fibrin/fibrinogen degradation products in superficial bladder cancer. Urol Clin North Am 2000; 27:39–46.[CrossRef][Medline]
  43. Morgan RO, Fernandez MP. Annexin gene structures molecular evolutionary genetics. Cell Mol Life Sci 1997; 53:508–15.[CrossRef][Medline]
  44. Evans TC, Nelsestuen GL. Calcium and membrane-binding properties of monomeric and multimeric annexin II. Biochemistry 1994; 33:13231–38.[CrossRef][Medline]
  45. Emans N, Gorvel JP, Walter C, Gerke V, Kellner R, Griffiths G, Gruenberg J. Annexin II is a major component of fusogenic endosomal vesicles. J Cell Biol 1993; 120:1357–69.[Abstract/Free Full Text]
  46. Creutz CE. The annexins and exocytosis. Science 1992; 258:924–31.[Abstract/Free Full Text]
  47. Chung CY, Erickson HP. Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-c. J Cell Biol 1994; 126:539–48.[Abstract/Free Full Text]
  48. Hajjar KA, Jacovina AT, Chacko J. An endothelial cell receptor for plasminogen/tissue plasminogen activator I. Identity with annexin II. J Biol Chem 1994; 269:21191–97.[Abstract/Free Full Text]
  49. Tokes AM, Hortovanyi E, Kulka J, Jackel M, Kerenyi T, Kadar A. Tenascin expression and angiogenesis in breast cancers. Pathol Res Pract 1999; 195:821–28.[Medline]



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