Plasmin-Cleaved β-2-Glycoprotein 1 Is an Inhibitor of Angiogenesis
2007; Elsevier BV; Volume: 171; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.070146
ISSN1525-2191
AutoresTaro Sakai, Krishnakumar Balasubramanian, Sourindra N. Maiti, Jyotsna B. Halder, Alan J. Schroit,
Tópico(s)Platelet Disorders and Treatments
Resumoβ-2-Glycoprotein 1, an abundant plasma glycoprotein, binds anionic cell surfaces and functions as a regulator of thrombosis. Here, we show that cleavage of the kringle domain at Lys317/Thr318 switches its function to a regulator of angiogenesis. In vitro, the cleaved protein specifically inhibited the proliferation and migration of endothelial cells. The protein was without effect on preformed endothelial cell tubes. In vivo, the cleaved protein inhibited neovascularization into subcutaneously implanted Matrigel and Gelfoam sponge implants and the growth of orthotopically injected tumors. Collectively, these data indicate that plasmin-cleaved β-2-glycoprotein 1 is a potent antiangiogenic and antitumor molecule of potential therapeutic significance. β-2-Glycoprotein 1, an abundant plasma glycoprotein, binds anionic cell surfaces and functions as a regulator of thrombosis. Here, we show that cleavage of the kringle domain at Lys317/Thr318 switches its function to a regulator of angiogenesis. In vitro, the cleaved protein specifically inhibited the proliferation and migration of endothelial cells. The protein was without effect on preformed endothelial cell tubes. In vivo, the cleaved protein inhibited neovascularization into subcutaneously implanted Matrigel and Gelfoam sponge implants and the growth of orthotopically injected tumors. Collectively, these data indicate that plasmin-cleaved β-2-glycoprotein 1 is a potent antiangiogenic and antitumor molecule of potential therapeutic significance. 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Indeed, recent studies raised the possibility that nβ2GP1 functions as an antiangiogenic molecule in vivo.34Beecken WDC Engl T Ringel EM Camphausen K Michaelis M Jonas D Folkman J Shing Y Blaheta RA An endogenous inhibitor of angiogenesis derived from a transitional cell carcinoma: clipped β2-glycoprotein-I.Ann Surg Oncol. 2006; 13: 1241-1251Crossref PubMed Scopus (21) Google Scholar It this article, we demonstrate that nβ2GP1 inhibits EC proliferation in vitro, inhibits neovascularization into subcutaneously implanted Matrigel and Gelfoam plugs, and blocks tumor growth in a mouse model system. Taken together, these data provide evidence in support of the concept that nβ2GP1 plays a regulatory role in EC physiology and angiogenesis. Male C57Bl/6 and BALB/c mice were purchased from the National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD). Tramp C2RE3 prostate adenocarcinoma cells (TRAMP) were provided by J. Killion, M. D. Anderson Cancer Center. These cells were derived from TRAMP C3 cells35Foster BA Gingrich JR Kwon ED Madias C Greenberg NM Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model.Cancer Res. 1997; 57: 3325-3330PubMed Google Scholar by selection for aggressively growing tumors after repeated orthotopic injections. The cells were maintained in vitro in minimal essential media containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA). Bovine aortic endothelial cells (BAECs) were cultured in bovine endothelial growth medium (Cell Applications, Inc., San Diego, CA). Human umbilical vein endothelial cells (HUVECs) were cultured in Medium 200 containing low serum growth supplement (Cascade Biologicals, Portland, OR). Plasmin and its chromogenic substrate, S-2251, were purchased from Chromogenix (Lexington, MA). Human serum albumin (HSA) was from Alpha Therapeutics (Los Angeles, CA), and annexin 2 antibodies (clone 5) and Matrigel were from BD Biosciences (Bedford, MA). Mouse CD31 antibodies (clone CO.3R1D4) were from Serotec (Raleigh, NC). Other chemicals and chromatographic media were from Sigma-Aldrich (St. Louis, MO). Rabbit anti-human angiostatin (Oncogene Research Products, San Diego, CA) exhibited significant cross-reactivity with plasminogen and plasmin. Proteins used in this study were routinely tested to ensure the absence of lipopolysaccharide contamination using the Pyrochrome LAL reagent (Associates of Cape Cod Inc., East Falmouth, MA) assay. Immobilized plasmin was prepared by incubating 1 mg of plasmin in ice-cold PBS with 1 ml of Affi-Gel 10 (Bio-Rad Laboratories, Hercules, CA). The coupling was allowed to proceed at 4°C for 2 hours, after which uncoupled reagents were removed by repeated washings with PBS. Polyclonal antibodies to iβ2GP1 were produced in rabbits by multiple intradermal injections of 0.5 mg of β2GP1 in complete Freund's adjuvant in multiple intradermal sites, followed by two boosters (0.25 mg of protein) at 2-week intervals in incomplete Freund's adjuvant. The rabbits were bled 2 weeks after the last injection. IgG was purified from the immune serum by protein G affinity chromatography. Syngeneic mouse red blood cells were labeled with 51Cr by incubation at 37°C for 4 hours with 0.25 mCi of Na51chromate (GE Healthcare, Little Chalfont, Buckinghamshire, UK) in Hepes-buffered saline (pH 7.4) containing 30 mmol/L glucose. Unbound 51Cr was removed by repeated washings with the same buffer. The cells were resuspended to a 25% hematocrit in the same buffer before injection. Intact β2GP1 was purified from pooled human plasma as described previously.36Polz E Kostner GM The binding of beta 2-glycoprotein-I to human serum lipoproteins: distribution among density fractions.FEBS Lett. 1979; 102: 183-186Crossref PubMed Scopus (194) Google Scholar, 37Wurm H β2-Glycoprotein-I (apolipoprotein H) interactions with phospholipid vesicles.Int J Biochem. 1984; 16: 511-515Crossref PubMed Scopus (222) Google Scholar In brief, whole blood collected from healthy volunteers (Gulf Coast Regional Blood Center, Houston, TX) was centrifuged at 2500 × g for 10 minutes to sediment the blood cells. The supernatant (plasma) was then chilled on ice, and perchloric acid [1.5% (v/v)] was added dropwise with continuous stirring. The plasma was incubated on ice for 15 minutes, followed by centrifugation at 20,000 × g for 15 minutes to sediment the precipitated proteins. The supernatant containing β2GP1 was brought to pH 7.0 with saturated sodium bicarbonate and dialyzed against Tris buffer (50 mmol/L Tris, pH 8.0) containing 20 mmol/L NaCl. The dialysate was passed over a DEAE-Sephacel column equilibrated with the same buffer. The flow-through was collected and passed over a Hi-Trap Heparin-Sepharose affinity column. The column was washed with Tris buffer containing 20 mmol/L NaCl, and the bound β2GP1 was eluted with the same buffer containing 250 mmol/L NaCl. Purity was assessed by gel electrophoresis and Western blotting with rabbit anti-human iβ2GP1. The identity of the protein was confirmed by N-terminal sequencing. Intact β2GP1 was incubated with immobilized plasmin at 37°C for 17 hours. The beads were removed by centrifugation and the supernatant recovered. Cleavage was verified by an electrophoretic shift under reducing conditions and by N-terminal sequencing, which revealed a second N terminus corresponding to the Lys317/Thr318 cleavage site. Western blotting of the purified product indicated that the nβ2GP1 preparations were plasmin-free and did not contain autoproteolytic products (no reactivity with plasmin or angiostatin antibodies). BAECs and TRAMP cells were incubated with iβ2GP1 or nβ2GP1 (4 μmol/L) on ice for 30 minutes. The cells were then washed with PBS and incubated for an additional 30 minutes on ice with 2 μg of biotinylated rabbit anti-human β2GP1 IgG, followed by incubation with 50 ng of fluorescein isothiocyanate (FITC)-streptavidin. Binding was determined by flow cytometric analysis using cells incubated only with the primary antibody and FITC-streptavidin as negative controls. For the competition experiments with annexin 2 antibody, BAECs were cultured on glass coverslips for 24 hours and incubated on ice for 1 hour with iβ2GP1 or nβ2GP1 (4 μmol/L) in the absence or presence of annexin II or CD31 (negative control) antibodies (0.33 μmol/L). The cells were then washed, fixed with 2% paraformaldehyde, and stained with biotinylated rabbit anti-human β2GP1 IgG (2 μg), followed by phycoerythrin-conjugated streptavidin (100 ng). BAECs or HUVECs were cultured to 80% confluence. One milliliter of conditioned or fresh (negative control) medium was transferred to cuvettes, and the change in absorbance at 405 nm was recorded following the addition of the chromogenic plasmin substrate S-2251 (0.3 mmol/L). BAECs and TRAMP C2RE3 cells were cultured in complete medium containing 0.5 μCi of [3H]thymidine and 4 μmol/L HSA (control), iβ2GP1, or nβ2GP1. After 72 hours, the cells were washed three times with PBS, twice with 5% trichloroacetic acid, and solubilized in 0.2% SDS. The cell lysate was resuspended in 5 ml of scintillation cocktail for liquid scintillation counting. HUVECs were cultured in 96-well plates in medium containing 4 μmol/L HSA (control), iβ2GP1, or nβ2GP1 (4 μmol/L). After 72 hours, 25 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (2.5 mg/ml) was added to each well and incubated for 2 hours at 37°C. The medium was then removed and the formazan crystals solubilized in 50 μl of dimethyl sulfoxide before spectrophotometric quantification (A = 560 nm). Cell proliferation was expressed as the percentage of controls. Cells were plated at ∼70% confluency on 6.5-mm Transwell polycarbonate membranes (8-μm pore size; Corning, Acton, MA). Vascular endothelial growth factor (VEGF) (25 ng/ml) was added to the lower chamber, and iβ2GP1 or nβ2GP1 (4 μmol/L) was added to the upper chamber. After 5 hours at 37°C, the cells on the upper surface were removed by scraping. The polycarbonate filters were then stained with Hema-Diff reagent (StatLab Medical Products, Inc. Lewisville, TX). Results are expressed as the mean ± SD of 10 individual experiments. Cells were cultured on 24-well tissue culture plates to confluency. Cells in the center of the wells were removed by scratching with a 1-ml pipette tip.38Kim JS Yu HK Ahn JH Lee HJ Hong SW Jung KH Chang SI Hong YK Joe YA Byun SM Lee SK Chung SI Yoon Y Human apolipoprotein(a) kringle V inhibits angiogenesis in vitro and in vivo by interfering with the activation of focal adhesion kinases.Biochem Biophys Res Commun. 2004; 313: 534-540Crossref PubMed Scopus (41) Google Scholar The remaining adherent cells were washed twice with PBS, incubated with iβ2GP1 or nβ2GP1 (4 μmol/L) in BAEC medium for 8 hours, and photographed. Motility was measured by counting the number of cells that repopulated the cleared area. Results are expressed as the number of cells/mm2 ± SD and are the mean of four individual experiments. Forty-eight-well tissue culture plates were coated with 250 μl of Matrigel (8.9 mg/ml) for 2 hours at 37°C. Cells (7.5 × 104) were plated in BAEC medium for 96 hours to allow for tube formation. The preformed tubes were then incubated with HSA, iβ2GP1, or nβ2GP1 (4 μmol/L) for 24 hours and assessed for tube integrity by microscopy. The effect of iβ2GP1 and nβ2GP1 on neovascularization was determined by two independent assays. Sterile Gelfoam absorbable sponges (Pharmacia & Upjohn, Peapack, NJ) were cut into 5 × 5 × 7-mm pieces and hydrated overnight with PBS. Agarose (0.4%, 100 μl) containing VEGF (2 pmol/implant) and nβ2GP1 (0.2 μmol) or HSA (0.2 μmol, control) was pipetted onto each sponge. After 1 hour at room temperature, the gel foams were placed into a subcutaneous pocket as described previously.39McCarty MF Baker CH Bucana CD Fidler IJ Quantitative and qualitative in vivo angiogenesis assay.Int J Oncol. 2002; 21: 5-10PubMed Google Scholar Vascularization into the implants was quantified after 2 weeks by assessing blood volume after i.v. injection of 51Cr-labeled syngeneic red blood cells several minutes before recovery of the implants. Blood volume was calculated from the specific activity of the blood (cpm/μl blood/g implant). Matrigel (1.5 ml) was mixed on ice with VEGF (0.7 pmol) in the presence or absence of iβ2GP1 or nβ2GP1 (6 nmol). BALB/c mice (three per group) were injected intradermally with 0.5 ml of the Matrigel. Two weeks later, 51Cr-labeled syngeneic red blood cells were injected i.v. several minutes before recovery of the implants. Blood volume was calculated as described for the gel foams. TRAMP C2RE3 cells (2 × 104) were implanted orthotopically into the prostate of 6-week-old, C57Bl/6 mice. The mice were randomly assigned to different groups. nβ2GP1 or iβ2GP1 (1.7 mg/0.2 ml pump) was administered to the mice with Alzet 2002 mini-osmotic pumps (delivery rate of 3.6 mg/kg/day; Durect Corp., Cupertino, CA) that were implanted i.p. and s.c. on days 1 and 14, respectively (spent pumps were not removed). Mice in the chemotherapy and combination therapy groups were also administered docetaxel intraperitoneally at 8 mg/kg once a week for 4 weeks beginning on day 3. Animals were sacrificed on day 28, and the tumors were harvested, weighed, and quick-frozen for immunohistochemistry. Frozen sections were stained for CD31-positive EC with rat anti-CD31 antibody (BD Biosciences) followed by Texas Red-conjugated goat anti-rat IgG (Jackson ImmunoResearch). Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining performed according to manufacturer's (Promega Corporation, Madison, WI) instructions. BAECs were grown for 72 hours in the absence or presence of intact or nβ2GP1 (4 μmol/L). The supernatants were centrifuged to remove cell debris and incubated with rabbit anti-human β2GP1. The antibody and bound antigens were concentrated by pull-down with protein G-Sepharose beads. The beads were washed and solubilized with SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer and resolved by gel electrophoresis. For Western blotting, the proteins were transferred to polyvinylidene difluoride membranes and probed with the same antibodies followed by peroxidase-conjugated anti-rabbit IgG. One microgram of Glu-plasminogen, plasmin, or angiostatin was incubated with 10 μg of iβ2GP1 or nβ2GP1 for 1 hour at 20°C, followed by incubation for 1 hour with control rabbit IgG or rabbit anti-β2GP1 IgG (20 μg) and protein G-Sepharose (20 μl). Captured IgG (and bound proteins) were centrifuged, and the supernatant (unbound protein) was mixed with SDS-PAGE sample buffer for Western blot analysis. The protein G beads were washed twice with PBS, centrifuged through 30% sucrose in PBS, and resuspended in SDS-PAGE sample buffer. Intact β2GP1 was purified from pooled human plasma by perchloric acid treatment followed by ion exchange and heparin affinity chromatography as described in Materials and Methods. SDS-PAGE and Western blot analysis showed that the purity of the protein was >98% (Figure 1). Nicked β2GP1 was prepared by incubating iβ2GP1 for 17 hours at 37°C with immobilized plasmin. SDS-PAGE analysis of the product under reducing conditions showed that >98% of the protein was cleaved (Figure 1A). N-Terminal sequencing revealed that the protein was cleaved at amino acids 317 and 318 (Lys-Thr). Western blotting of the final preparation with plasmin antibodies indicated that the nβ2GP1 was plasmin-free. Solid-phase enzyme-linked immunosorbent assay binding assays were performed to determine the binding of the nicked isoform to anionic phosphatidylserine (PS). Figure 1B shows that the ability of iβ2GP1 to bind PS was lost after plasmin treatment. Control experiments showed that neither isoform bound neutral phosphatidylcholine. Previous studies have shown that iβ2GP1 binds to the surface of EC through annexin 2 expressed at the cell surface.24Ma K Simantov R Zhang JC Silverstein R Hajjar KA McCrae KR High affinity binding of beta 2-glycoprotein I to human endothelial cells is mediated by annexin II.J Biol Chem. 2000; 275: 15541-15548Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar To determine the binding of iβ2GP1 and nβ2GP1, BAECs were incubated with the proteins for 30 minutes. Fluorescence-activated cell sorting analysis after incubation of the cells with biotinylated anti-human-β2GP1 followed by FITC-conjugated streptavidin showed that both iβ2GP1 and nβ2GP1 bound to >75% of the EC (Figure 2). Unlike EC, only the intact protein bound to TRAMP C2RE3 cells. Several studies were performed to determine the influence of iβ2GP1 and nβ2GP1 on EC function. First, the effect of β2GP1 on EC migration was determined by assessing cell mobility using the Boyden chamber transwell migration assay. Figure 3, A and B, shows that nβ2GP1, but not iβ2GP1, significantly inhibited the migration of cells to the lower chamber. Additional evidence supporting this observation was obtained with the in vitro wound healing model system.38Kim JS Yu HK Ahn JH Lee HJ Hong SW Jung KH Chang SI Hong YK Joe YA Byun SM Lee SK Chung SI Yoon Y Human apolipoprotein(a) kringle V inhibits angiogenesis in vitro and in vivo by interfering with the activation of focal adhesion kinases.Biochem Biophys Res Commun. 2004; 313: 534-540Crossref PubMed Scopus (41) Google Scholar Confluent EC monolayers incubated with iβ2GP1 or nβ2GP1 were scraped, and migration of cells into the denuded sections was monitored at various time intervals. Figure 3, C and D, shows that, whereas control and iβ2GP1-treated ECs repopulated the denuded area within 8 hours, incubation in the presence of nβ2GP1 resulted in ∼60% inhibition in cell migration. Control experiments showed that nβ2GP1 did not inhibit migration of TRAMP cells, suggesting that the inhibitory effect of nβ2GP1 was EC-specific (Figure 3C). In contrast to the apparent specific effect of nβ2GP1 on BAEC migration, both iβ2GP1 and nβ2GP1 significantly inhibited BAEC proliferation (Figure 4A). Growth of TRAMP C2RE3 cells (and PC3, LnCap, MCF7, MDA-MB-231, and MDA-MB-435 cells, data not shown) was unaffected when incubated with either protein (Figure 4C). In contrast to BAECs, HUVECs were sensitive to the inhibitory effect of the nβ2GP1, but not iβ2GP1 (Figure 4B).Figure 4β2GP1 inhibits EC proliferation. BAECs (A), HUVECs (B), or TRAMP C2RE3 (C) cells were cultured for 72 hours in complete medium in the presence of HSA (control), iβ2GP1, or nβ2GP1. Cell proliferation was assessed by [3H]thymidine incorporation (A and C) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (B) as described in Materials and Methods. R
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