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Tumor Biology |
CuraGen Corp., Branford, Connecticut 06405
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A delicate balance between positive and negative regulatory signals controls each step of angiogenesis (6 , 7) . VEGF and bFGF are two of the best characterized angiogenesis agonists (8) . Among the several types of angiogenesis inhibitors are proteolytic fragments of Type XVIII collagen and plasminogen, named endostatin and angiostatin, respectively (9, 10, 11) . Systemic administration of recombinant endostatin and angiostatin caused growth regression of a number of tumor types in murine xenograft models (10 , 12) . Moreover, in preclinical animal efficacy models, administration of endostatin can lead to stable tumor dormancy without the development of acquired drug resistance (12) . Currently, endostatin and angiostatin are being evaluated in clinical trials for a variety of human malignancies. Other antiangiogenic factors that have been evaluated preclinically include platelet factor-4 (13) , thrombospondin-2 (14) , the Mr 16,000 NH2-terminal fragment of prolactin (15) , and maspin (16) .
The recent discovery of angiopoietins has provided additional insight into the molecular and cellular mechanisms of blood vessel formation (4
, 17)
. Angiopoietins (Ang 1 and Ang 2) are Mr 
70,000 proteins that share considerable sequence homology. Each protein consists of a signal peptide, an NH2-terminal coiled-coil domain, a short linker peptide region, and a COOH-terminal FD. The coiled-coil region is responsible for dimerization of angiopoietin, and the FD binds to Tie2 receptors. Both Ang 1 and Ang 2 form dimers and oligomers (18
, 19)
. In vivo analysis by targeted gene disruption reveals that Ang 1 recruits and sustains periendothelial support cells (4
, 18
, 20)
, whereas Ang 2 disrupts blood vessel formation in the developing embryo by antagonizing the effect of Ang 1 on the Tie2 receptor (17)
. Later, Ang 4 was shown to be a third protein capable of binding to the Tie2 receptor (21)
. Three additional proteins (ARP1, ARP2, and CDT6) with similarity to angiopoietins have also been discovered (22, 23, 24, 25)
that do not bind to the Tie2 or Tie1 receptor and do not possess a specific cysteine motif that is characteristic of angiopoietins. The molecular mechanism through which these proteins regulate angiogenesis has not been fully elucidated (21)
.
Here, we used a homology-based gene mining approach to isolate an angiopoietin-related cDNA identical to ARP-1 that we designate as angioarrestin, because of its antiangiogenic properties both in vitro and in vivo. The mRNA expression profile of angioarrestin was strikingly down-regulated in tumor tissue in comparison to matched NAT. To functionally characterize angioarrestin, the cDNA was expressed in a eukaryotic expression system, and recombinant protein was purified. Systematic analysis in a variety of cell-based angiogenesis assays revealed for the first time that angioarrestin inhibited a number of angiogenic processes, including proliferation, migration, tube formation, and endothelial cell adhesion. These effects were endothelial cell specific and not observed in nonendothelial cells. In additional studies, nude mice were injected with HT1080 tumor cells engineered to ectopically express angioarrestin. Compared with control, these mice had a marked reduction in number and size of tumor nodules. In summary, these results demonstrate a novel function for angioarrestin as an inhibitor of angiogenesis and indicate its potential utility as a cancer therapeutic.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation and Cloning of Human Angioarrestin.
A technology, SeqCalling, that provides DNA sequence information for the coding regions of expressed genes was used to identify different fragments encoding angioarrestin cDNA. Briefly, a partial angioarrestin cDNA was identified using a homology search of CuraGen’s SeqCalling database. The SeqCalling database was developed using a modification of the GeneCalling technology (26)
, except that digested fragments were size fractionated by high-performance liquid chromatography, PCR amplified, cloned, and then sequenced. All sequences were then collected into a database and collapsed into single gene assemblies based on sequence identity. Angioarrestin cDNA ends were generated by rapid amplification of cDNA ends using a cDNA pool from 28 separate tissues. On the basis of the predicted sequence, oligonucleotide primers were designed to amplify full-length angioarrestin cDNA by PCR. The primers used were as follows: forward primer, 5'-CCA CCA TGA AGA CTT TTA CTT TTA CCT GGA CCC-3'; reverse primer, 5'-GTC AAT AGG CTT GAT CAT CAT CTG AAC TG-3'. The PCR mix contained 4 µg of human heart cDNA, 75 pmol of primers, 5 µmol of deoxynucleotide triphosphates, 1 unit of Fidelity expand polymerase, and 5 µl of Fidelity expand buffer (Boehringer Mannheim). A touchdown PCR was used as per standard protocol (27)
. A single PCR product of 1.4 kb was obtained and cloned into pcDNA3.1V5his Topo vector (Invitrogen). A Hind III-Pme I fragment from pcDNA3.1/angioarrestin was ligated into pCEP4, and the resulting vector was named as pCEP4/angioarrestin. This vector contains an in-frame V5 and His6 tag at the 3'-terminus of the coding region.
Expression, Purification, and Biochemical Characterization of Recombinant Angioarrestin.
The pCEP4/angioarrestin vector was transfected into HEK293T cells using Lipofectamine Plus (Life Technologies, Inc., Rockville, MD). The conditioned medium was collected from transfected cells after 72 h, pooled, and loaded onto a Ni2+ affinity column (Qiagen, Valencia, CA). The column was washed with PBS (pH 7.4), containing 500 mM NaCl, followed by the same buffer containing 5 mM imidazole. The bound protein was eluted with PBS (pH 7.4), containing 500 mM imidazole, pooled, and dialyzed overnight in PBS (pH 7.4). The protein was further purified by a second round of purification over a Ni2+ affinity column and dialyzed against PBS (pH 7.4). Protein concentration was determined using the Bradford reagent (Bio-Rad, Hercules, CA). Molarity was calculated using the molecular weight of the dimer. Protein purity was assessed by Silver staining after SDS-PAGE analysis using a 4–15% Tris/glycine gradient gel. Western blot analysis was performed with anti-V5 tag mAb (1:5000; Ref. 28
) conjugated to horseradish peroxidase, followed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Angioarrestin was tested for endotoxin using the Gel Clot method (Cape Cod Associates, Cape Cod, MA) and found to have <10 EU/mg protein. Purified protein was used in all in vitro assays.
RTQ-PCR Expression Analysis.
Human normal, tumor, or matched normal adjacent tumor tissues were obtained from National Cancer Institute’s Cooperative Human Tissue Network or The National Disease Research Institute. Tumor cell lines were derived from cultured cells obtained from American Type Culture Collection. RNA samples were prepared from the tissues using TRIzol reagent according to the manufacturer’s instructions (Life Technologies, Inc.). RTQ-PCR (29)
was performed on an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA) using TaqMan reagent (PE Applied Biosystems). RNAs were normalized using human ß actin and glyceraldehyde-3-phosphate dehydrogenase according to the manufacturer’s instructions. Equal quantity of normalized RNA was used as a template in PCR reactions with angioarrestin-specific reagents to obtain threshold cycle values. The following angioarrestin-specific primers and probe were used: forward primer 5'-GCT GGG AGG TAA CGA GAT TCA G-3', probe primer Fam 5'-ATC CAG GTT ATC CCA GAG ATT TAA TGC CAC C-3' Tamra, and reverse primer 5'-TGG GAG AAG TTG CCA GAT CAG-3'.
Endothelial Cell BrdUrd Incorporation Assay.
The effect of angioarrestin on proliferation was assessed using HUVEC, HMVEC-d, and C-PAE. The cells were plated in 96-well flat-bottomed plates precoated with Attachment Factor (Cascade Biologics) at 3 x 104 cells/well in 100 µl of Medium 200 (Cascade Biologics) containing 0.5% FBS. After 24 h of starvation at 37°C, the cells were washed two times with serum-free medium and then fed with fresh medium containing 1% FBS with VEGF165 and bFGF (10 ng/ml; R&D Systems, Minneapolis, MN) with and without angioarrestin protein. The BrdU assay was performed according to the manufacturer’s specification (Roche Molecular Biochemicals, Indianapolis, IN).
Migration Assay.
To determine the ability of recombinant angioarrestin to block migration of HUVEC and HMVEC-d toward VEGF165, 24-well transwell (BD Biosciences, Bedford, MA) migration chambers having an 8-µm pore size were used. The transwells were coated with 10 µg/ml Type I collagen (BD Biosciences) from rat tail for 1 h at 37°C. After washing with PBS, the wells were seeded with HUVEC suspended at 2 x 105 cells/ml in Medium 200 containing 1% BSA (Sigma Chemicals, St. Louis, MO). The bottom chambers (600 µl) were filled with Medium 200 containing 1% FBS supplemented with 10 ng/ml recombinant VEGF165. The top chamber was seeded with 4 x 104 cells/well in 200 µl containing different angioarrestin concentrations. Cells were allowed to migrate for 4 h at 37°C. After incubation, cells on the top surface of the membrane (nonmigrated cells) were scraped with a cotton swab. Cells on the bottom side of the membrane (migrated cells) were stained with 0.2% Crystal Violet dye (Fisher Scientific, Springfield, NJ) in 70% ethanol for 30 min. The cells were then destained in PBS (pH 7.4), and the membrane was left to air dry at room temperature. Migrated cells were counted using a Zeiss Axiovert 100 inverted microscope. Three independent areas per filter were counted, and the mean number of migrated cells was calculated. RGD control peptide (Invitrogen; Cat. No. 12135-018) having the sequence GRGDSP was used as a positive control. Each experiment was done in duplicate and repeated four times.
Endothelial Cell Tube Formation Assay.
Endothelial tube formation assays using HUVEC and HMVEC-d were performed as described previously (30)
. Briefly, HUVECs were trypsinized, washed with PBS (pH 7.4), counted, and resuspended in Medium 200 containing 2% FBS supplemented with 10 ng/ml VEGF165 in the presence or absence of different concentrations of angioarrestin. Cells (25,000) were seeded per well onto solidified Matrigel (BD Biosciences) in 96-well plates. The cells were incubated at 37°C for 18–20 h. Tubular networks were visualized with an inverted phase contrast Zeiss Axiovert 100 inverted microscope. Each dilution was done in duplicate, and the assay was repeated three times. The tubular network patterns were captured using a Kodak digital camera and analyzed using a PhotoShop 5.5 program.
Endothelial Cell Adhesion Assay.
Untreated 96-well flat-bottomed tissue culture plates (Fisher Scientific) were used in the cell adhesion assay. The plates were coated with 10 µg/ml different ECM proteins (Type I collagen, Type IV collagen, fibronectin, vitronectin, laminin, and Matrigel) overnight at 4°C. The remaining protein binding sites were blocked with 1% BSA in PBS (pH 7.4) for 2 h at 37°C. HUVECs were grown to subconfluence (70–80%) in Medium 200. The cells were labeled with Calcein-AM fluorophore (Molecular Probes, Eugene, OR) as described (31)
. The cells were trypsinized, washed, and resuspended at 1.5 x 105 cells/ml in serum-free medium containing 1% BSA. The cells were then mixed with different concentrations of angioarrestin in 100-µl volumes containing 2 x 104 cells/treatment for 15 min at room temperature. After incubation, the cell suspension was then added to each well, and the plates were incubated at 37°C for 45 min in 5% CO2. At the end of the incubation period, unattached cells were removed by washing three times with serum-free medium, and attached cells were counted using a Cytofluor 4000 fluorometer (PE Applied Biosystems). The number of attached cells was represented as a percentage of endothelial cell adhesion, as determined by the ratio of attached cells in the presence or absence of factor.
Inhibition of Tumor Nodule Formation.
The plasmid containing angioarrestin (pcDNA3/angioarrestin) was used to transfect HT1080 cells with Lipofectamine Plus reagent according to the manufacturer’s protocol. Cells were supplemented with 10% FBS 5 h after transfection. The transfectants were grown in DMEM (Life Technologies, Inc.) containing 10% FBS supplemented with 500 µg/ml G-418 (Life Technologies, Inc.), and angioarrestin expression was confirmed by Western blot analysis. As control, HT1080 cells were transfected with the appropriate empty vector and selected as described above. After 2 weeks of culture, pools of transfected cells were subcultured, washed with PBS (pH 7.4), and used as described below.
Athymic nude mice (five per group) were injected i.v. with 0.5 x 106 cells of HT1080 control pool or HT1080 angioarrestin in a volume of 200 µl. Four weeks after injection, the animals were sacrificed, and lung tissues were harvested and infused with formalin. The entire lung tissues were embedded, and step sections (400 µm) were prepared and stained with H&E using a standard protocol (O’Reilly et al., 1994a). The tumor number and area were assessed by morphometric analysis on three stepwise sections from each animal. For calculating mean tumor area (µm2), the length and width of the tumor were microscopically measured using an ocular µm.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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encodes a protein of 491 amino acids that was found to be 100% identical to ARP-1. Because of its antiangiogenic properties in both the in vitro and in vivo analysis detailed below, we propose changing the name of ARP-1 to angioarrestin. Angioarrestin has 58.6, 44.7, 31.9, 31.0, and 29.4% overall amino acid identity to ARP2, CDT6, Ang 1, Ang 2, and Ang 4, respectively. Like all angiopoietins or related proteins, angioarrestin has a signal peptide, coiled-coil region, and a FD. However, angioarrestin has only one of three closely spaced conserved cysteines found in the FD of angiopoietin family members (21)
. Genomic DNA analysis showed that angioarrestin is localized to human chromosome 1 (1q24.2) and contains four exons.
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and is most abundant in adrenal gland, placenta, and small intestine. Interestingly, a unique expression profile of angioarrestin was noted in tumor and matched NAT (Fig. 2B)
. These data demonstrate that tumor tissue from prostate, lung, kidney, thyroid, and bladder cancer showed dramatically less expression when compared with normal adjacent tissue.
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60,000 and 64,000 under reducing conditions (Fig. 3A
, Lane 2). In addition, a minor species was observed at Mr 120,000. Under nonreducing conditions (Fig. 3A
, Lane 3), a predominant protein of Mr 120,000 was observed along with minor higher molecular weight products. Proteins of corresponding molecular weight were also detected by Western blot analysis with a mAb that detects the V5 tag engineered in the COOH terminus of angioarrestin (Fig. 3B
, Lanes 1 and 2). These results suggest that angioarrestin exists predominantly as a disulfide-linked dimer.
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, angioarrestin inhibited VEGF/bFGF-induced BrdUrd incorporation in a dosedependent manner. The IC50 was 75, 90, and 250 ng/ml (0.6, 0.8, and 2 nM) for HUVEC, HMVEC-d, and C-PAE, respectively. Angioarrestin did not inhibit the bFGF-stimulated proliferation of nonendothelial cells, such as NIH 3T3 and CCD1070 (data not shown). These results suggest that angioarrestin inhibits the proliferation of various endothelial cells derived from different vessel types.
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. Inhibition (68%) observed with the highest dose (100 ng/ml, 0.8 nM) tested was comparable with that seen with a control RGD peptide (50 and 70% inhibition at 1 and 10 µg/ml, respectively; data not shown). No effect was seen on nonendothelial cell lines (NIH 3T3 and CCD1070 treated with bFGF) at angioarrestin concentrations 
10 µg/ml (data not shown). These results demonstrate that angioarrestin inhibits the VEGF-mediated migration of endothelial cells.
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. A dose-dependent inhibition of tubular network formation was observed in the range of 1–10 µg/ml (8–80 nM) angioarrestin. A similar dose response was seen when HMVEC-d were used for tube formation (data not shown). Thus, angioarrestin inhibits tube formation in large and small vessel endothelial cells.
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. At 10 µg/ml (80 nM), there was a 58% reduction of HUVECs in binding to fibronectin-treated plates. Similar inhibitory profiles were also observed with collagen (Type I and IV) and laminin ECM proteins (data not shown). In addition, angioarrestin also inhibited adhesion of HMVEC-d with a similar inhibitory profile (Fig. 7B)
. Additional experiments demonstrated that angioarrestin did not induce apoptosis in adherent HUVECs over a 12–15 h treatment (data not shown). Together, these findings show that angioarrestin inhibits endothelial cell adhesion to different ECM proteins.
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. In addition, tumors from angioarrestin-transfected HT1080 cells were significantly smaller (P < 0.05) when compared with control (Fig. 8B)
. As shown in Fig. 8C
, tumor tissue from angioarrestin-transfected HT1080 cells had fewer blood vessels (arrows) that were markedly smaller and less mature than control. Thus, the ectopic expression of angioarrestin in HT1080 fibrosarcoma cells inhibited tumor nodule formation and decreased tumor size in comparison to control HT1080 transfectants, an effect that was likely attributable to an inhibition of tumor-associated angiogenesis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using a sensitive RTQ-PCR assay, angioarrestin mRNA was found to be lower in tumor tissues relative to matched NAT. Ample experimental and clinical correlates exist for suppression of antiangiogenic factors during tumor progression. This concept was illustrated when reconstitution of a wild-type p53 tumor suppressor in a BT549 breast carcinoma cell line resulted in secretion of an angiogenesis inhibitor (32) . Down-regulation of the retinoic acid receptor ß2, maspin, and E-cadherin genes have correlated with cancer progression and increased tumor invasion or metastasis (16 , 33 , 34) . Invasiveness, vessel density, and poor clinical outcome have been reported to statistically correlate with down-regulation of thrombospondin-1 in advanced cervical cancer (35) . Likewise, a significant stepwise decrease in maspin expression parallels breast cancer progression from ductal carcinoma in situ, to locally invasive cancer, and finally to lymph node metastasis (36) . Decreased expression of maspin in prostate cancer has also been reported to correlate inversely with the development of local recurrence or systemic tumor progression (37) . Additional experiments will be needed to conclusively demonstrate a tumor suppressive role for angioarrestin and whether the decrease in angioarrestin expression results in tumor formation.
Angioarrestin inhibits angiogenic processes through effects on both induction and resolution phases of the angiogenic cascade. In the induction phase of angiogenesis, angioarrestin inhibited BrdUrd incorporation, migration, and endothelial cell adhesion to ECM proteins. The activity of angioarrestin (IC50 = 0.8 nM) to block endothelial cell BrdUrd incorporation compares favorably with other antiangiogenic molecules, including angiostatin (IC50 = 140 nM) and endostatin (IC50 = 20 nM; Refs. 9
and 10
). Angioarrestin was also more potent (IC50 = 0.3 nM) in the migration assay when compared with published data for endostatin (IC50 = 3 nM; Refs. 9
and 10
) and angiostatin (IC50 = 10 nM; Ref. 11
). With respect to the resolution phase, angioarrestin inhibited endothelial cell tube formation at 80 nM, whereas previous reports (9
, 10)
have shown that angiostatin was active in this assay at 200 nM. The potent in vitro activity of angioarrestin across all phases of the angiogenic process was confirmed in vivo. Ectopic expression of angioarrestin in HT1080 cells suppressed tumor growth when compared with control. This activity was comparable with endostatin, and angiostatin tested in similar models (38
, 39) . Additional studies will be required to determine whether angioarrestin can suppress tumor formation when administered systemically or through gene therapy.
Two additional molecules related to angioarrestin, ARP2 and CDT6, with significant homology to angioarrestin have also been described (23 , 25) . The possibility exists that these three molecules comprise a family of antiangiogenic proteins. Although this is the first report demonstrating that angioarrestin/ARP1 possesses antiangiogenic properties, evidence suggests that CDT6 may also inhibit angiogenesis (24) . Surprisingly, the only activity (22) observed for ARP1 and ARP2 was a modest endothelial cell-sprouting activity. It is difficult to rationalize the observed effect on sprouting considering the potent antiangiogenic activity that is demonstrated by angioarrestin in the present study. Interestingly, none of the three angiopoietin-related molecules bind to Tie2, the receptor through which angiopoietins function, nor do they interact with the closely related Tie1 receptor (23 , 24) . This raises the possibility that angioarrestin, ARP2, and CDT6 all interact with unidentified receptor(s) on endothelial cells. The mechanism by which angioarrestin exerts its antiangiogenic properties is currently under investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 M. D. and W. J. L. contributed equally to this manuscript. 
2 To whom requests for reprints should be addressed, at CuraGen Corp., 322 East Main Street, Branford, CT 06405. Phone: (203) 871-4331; Fax: (203) 315-3301.
3 The abbreviations used are: ECM, extracellular matrix; VEGF, vascular endothelial growth factor; C-PAE, calf pulmonary arterial endothelial cell; NAT, nontumor adjacent tissue; FD, fibrinogen homology domain; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; bFGF, basic fibroblast growth factor; HMVEC-d, human microvascular endothelial cells from dermis; RTQ, real-time quantitative; mAb, monoclonal antibody.
Received 2/12/02. Accepted 4/29/02.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1ß1 and 2ß1 integrins. Proc. Natl. Acad. Sci. USA, 94: 13612-13617, 1997.This article has been cited by other articles:
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, HUVEC; 
, HMVEC-d; 
, C-PAE.
