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Definitions

• the present invention relates generally to the field of cell-cell interaction.
• the present invention discloses novel functions for vascular endothelial growth factor and type I collagen inducible protein (VCIP) in cell-cell interaction and intracellular signaling.
• VCIP vascular endothelial growth factor and type I collagen inducible protein
• Endothelial cells which line the walls of blood vessels, are able to promote both "homotypic' and 'heterotypic' cell-cell interactions. Such interactions are critical for angiogenesis, which proceeds through several distinct coordinated steps. Initially, endothelial cells that are contact inhibited or considered to be in the G 0 phase of the cell cycle become activated in response to an increase in local concentrations of angiogenic factors. Activated endothelial cells then locally secrete proteases to dissolve basement membranes, thereby allowing endothelial cells to detach from the vascular wall.
• the detached endothelial cells then send out cytoplasmic projections, migrate, elongate extensively and form cell-cell interactions. Eventually, endothelial cells enter the cell cycle and can either differentiate into tube- like structures, depending upon the presence of specific survival factors and extracellular matrix (ECM) components, or undergo apoptosis, which can disrupt angiogenesis.
• ECM extracellular matrix
• VEGF vascular endothelial growth factor
• bFGF basic fibroblast growth factor
• VEGF vascular endothelial growth factor
• VEGF and bFGF can act on endothelial cells either individually or in a coordinated manner to transduce extracellular signals into distinct cellular transcriptional responses.
• the specific roles of VEGF and its receptors in angiogenesis have been well documented. While most of these angiogenic cytokines directly regulate normal angiogenesis, unrestrained production of these factors can potentially deregulate cell-cell interactions, cell-matrix interactions and gene expression. Such deregulation may contribute to various vascular abnormalities, including the growth of solid tumors, cardiovascular disease and diabetic retinopathy.
• Activated endothelial cells detach from the endothelium and maintain cell- cell contact in order to survive; the absence of such cell-cell interactions can promote anoikis.
• Endothelial cells-mediated cell-cell interactions are also required for the recruitment of pericytes, as well as for the stabilization and maturation of blood vessels.
• Molecules that mediate cell-cell interactions include integrins and their ligands, VE-cadherin, PECAM- 1 (CD31), junctional adhesion molecules (JAM), VCAM- 1, selectins, claudins, Eph and Ephrins. These adhesion molecules are also involved in the assembly and formation of adherent and tight junctions, phenotypes that are closely associated with the formation of mature blood vessels and the segregation of arteries and veins.
• VEGF vascular endothelial growth factor
• type I collagen inducible protein DDBJ/EMBL/GenBank accession No. AF480883
• PAP2b phosphatidic acid phosphatase type 2b
• the prior art is deficient in uses of vascular endothelial growth factor and type I collagen inducible protein in cell-cell interaction and infracellular signaling as well as pathophysiological states.
• the present invention fulfills this long-standing need and desire in the art.
• vascular endothelial growth factor and type I collagen inducible protein also known as phosphatidic acid phosphatase 2b (PAP2b)
• PAP2b phosphatidic acid phosphatase 2b
• VCIP/PAP2b exhibits an Arg-Gly-Asp (RGD) cell adhesion sequence.
• RGD Arg-Gly-Asp
• Immunoprecipitation and fluorescence-activated cell sorting analyses demonstrated that VCIP-RGD is exposed to the outside of the cell surface. Retroviral transduction of VCIP induced cell aggregation/cell-cell interactions, modestly increased pl20 catenin expression and promoted activation of the Fak, Akt and GSK3 ⁇ protein kinases.
• VCIP binds to pl20catenin on endothelial cells, but not VE-cadherin or other Armadillo domain-containing proteins such as ⁇ -catenin or ⁇ -catenin (plakoglobin).
• VCIP immunocomplex prepared from E-cadherin-deficient SW480 cell line contained pl20catenin immunoreactivity, suggesting VCIP and pl20catenin interaction may be E cadherin-independent.
• a truncated VCIP without C-terminal cytoplasmic domain failed to coprecipitate pl20catenin.
• the present invention further provides evidence that expression of VCIP potentiates tumor growth and metastasis in athymic nude mice by recruiting endothelial cells and regulating tumor angiogenesis.
• Human colorectal adenocarcinoma (SW480) cells stably expressing various human VCIP/PAP2b cDNA constructs were generated. These SW480 cells were xenografted subcutaneously into nude mice, and the role of VCIP in tumor growth, angiogenesis, and metastasis was monitored for a period of 30-45 days. Metastatic foci formation at distance sites were determined by the presence of human ALU DNA repetitive sequence in mouse tissue. Confrol SW480 parental cells were tumorigenic but did not grow beyond 2 mm in size.
• SW480 cells expressing VCIP-RGD (wild-type) promoted aggressive tumor growth beyond 2 mm, accompanied by tumor neovasculature formation and induced metastases to brain, liver, and lung.
• Phosphatase inactive VCIP/PAP2b and delta-C-cyto mutants also promoted tumor growth and neovascularization, but did not support metastasis.
• This response was greatly diminished in SW480 cell expressing VCIP-RGE (mutant), as illusfrated by the lack of neovascularization and metastasis.
• anti-VCIP/PAP2b-RGD antibody also significantly inhibited bFGF- and VEGF-induced experimental angiogenesis.
• VCIP/PAP2b contains at least two distinct functional domains (i.e., phosphatase enzyme and RGD cell adhesion) and phosphatase function is not required for angiogenesis. Both domains were shown to act in synergy to potentiate tumor growth, angiogenesis and metastasis in an unprecedented manner. Thus, the present invention reveals VCIP/PAP2b as a novel potential target for anti-angiogenic, anti-cancer and anti-metastatic therapy.
• vascular endothelial growth factor and type I collagen inducible protein in a cell.
• the present invention also provides methods of treating a patient havmg a pathological condition resulted from integrin-mediated cell-cell interaction.
• the methods involve blocking the binding of integrin to cell surface VCIP by antibodies directed against a VCIP peptide comprising a RGD sequence or by a RGD-containing peptide derived from VCIP.
• the function of VCIP can be blocked by VCIP anti-sense oligonucleotides.
• blocking integrin binding to VCIP can be used to inhibit angiogenesis and the formation of capillaries in a patient.
• the present invention provides peptides derived from VCIP, vectors encoding such peptides and antibodies directed against such peptides.
• Figures 1 A-M show expression analysis and predicted amino acid sequence of VEGF and type I collagen inducible protein (VCIP).
• Figure 1A HUVECs were embedded into three-dimensional type I collagen gel in the presence of 20% adult human serum, lx ITS and stimulated with VEGF 165 (100 ng/ml). Total RNA was prepared at the indicated time points. Northern blots were hybridized with a fragment ofthe VCIP (clone- 33 A) cDNA probe. The transcript size (3.4 kb) is indicated on the right. The numbers at the bottom ofthe gel represent the fold increase in VCIP mRNA levels, as compared with untreated cells.
• Figure IB Ethidium bromide-stained agarose gel shows equivalent amounts of RNA used.
• Figure IC HUVECs were cultured as above and left untreated, the last lane shows cells treated with VEGF for 12 h as a positive control. Total RNA was isolated at indicated time points and subjected to VCIP northern blot analysis.
• Figure ID The blot shown in Figure IC was stripped and re-hybridized with a GAPDH probe to show that equal amounts of RNA were loaded across the lanes.
• Figure IE Poly(A) + RNA prepared from various human tissues was subjected to VCIP northern blot analysis by hybridization with the clone-33A cDNA probe.
• Figure IF The blot shown in Figure IE was re-hybridized with a GAPDH probe, showing that equal amounts of RNA were loaded across the lanes.
• Figure 1G Detection of VCIP mRNA by RT-PCR (20 cycles): monolayer endothelial cells were treated for 30 min with: (1) untreated, (2) bFGF (20 ng/ml), (3) VEGF 165 (100 ng/ml), (4) PMA (20 ng/ml), (5) bFGF (20 ng/ml) + VEGF (100 ng/ml), (6) bFGF (20 ng/ml) + PMA (20 ng/ml), (7) VEGF (100 ng/ml) + PMA (20 ng/ml) and (8) bFGF (20 ng/ml) + VEGF (100 ng/ml) + PMA (20 ng/ml).
• Figures 1H-J RT- PCR analysis of uPAR mRNA (Figure IH), ⁇ -actin mRNA ( Figure II) and GAPDH mRNA levels (Figure 1 J) were canied out under the same conditions as described in Figure 1G. All experiments were repeated at least three times, with similar results.
• Figure IK Inserts from six recombinant _phage clones are shown, and the relative position of clone 33A is indicated.
• Figure IL The complete predicted amino acid sequence of VCIP. The putative transmembrane segments are underlined. The N-linked glycosylation site is indicated by an asterisk.
• lipid phosphatase catalytic domain is indicated by bold letters and underlined, and the RGD motif is boxed.
• Figure IM The amino acid sequence of human VCIP (top) is ahgned with other proteins containing lipid phosphatase-like catalytic motifs.
• Figures 2A-C show expression of VCIP requires ⁇ 2 ⁇ l integrin ligation
• VEGF 165 treatment Endothelial cells were embedded in 3D type I collagen for 12 hr and incubated in the presence or absence of VEGF, bFGF, and the indicated antibodies: 20.0 ⁇ g/ml of purified anti- ⁇ l ⁇ l (TS2/7, ATCC), 20.0 ⁇ g/ml of purified anti- ⁇ 2 ⁇ l integrin
• Figures 3A-F show VCIP induction by growth factors and cytokines.
• Indicated monolayer cells were stimulated with various growth factors and cytokines for 6 h in media M199 containing 10% serum + lx ITS.
• the concentrations of cytokines used were optimized according to their ability to induce Erk2 phosphorylation in western blot analysis: VEGF 165 (100 ng/ml), EGF (20 ng/ml), bFGF (30 ng/ml), TNF- ⁇ (15 ng/ml),
• RNA was subjected to northern blot analysis by hybridization with the indicated probes.
• the uPAR northern blot was included as a control for the cytokines used.
• Ethidium bromide-stained gels show equal amounts of RNA used ( Figures 3C and F). Data shown are representative of those obtained in two or three separate experiments.
• Figures 4A-J show schematic diagrams of various recombinant cDNA constructs used in this study.
• Figures 4A-D pEGFP-based constructs.
• Figures 4E-G pLNCX2 retroviral constructs.
• Figures 4H-J pGST-fusion protein constructs. The relative positions of RGD and RGE within the constructs are indicated. HA indicates hemaglutinin epitope tags. All constructs are shown in the 5 '-3' orientation. Arrows indicate the direction of transcription.
• Figures 5A-C show VCIP is a cell surface antigen.
• HEK293 cells were transiently transfected with the indicated constructs and subjected to cell surface biotinylation. Cell extracts were then subjected to immunoblotting or immunoprecipitation.
• Figure 5A Anti-GFP immunoblotting.
• Figure 5B Immunoprecipitation with anti-GFP monoclonal antibodies followed by streptavidin-HRP immunoblotting.
• Figure 5C FACS analyses of HEK293 cells stably expressing the V (vector alone) or WT (wild-type) constructs. Both cells were stained with anti-VCIP- RGD mAb (30 ⁇ g/ml) followed by FITC-labeled anti-rabbit IgG antibodies. Data shown are representative of those obtained in at least three separate experiments.
• Figures 6A-K show VCIP promotes cell-cell interactions.
• Figure 6A HEK293 cells were retrovirally transduced with either pLNCX2 (V cells, vector only), pLNCX2-VCIP-RGD (WT cells, wild-type) or pLNCX2-VCIP-RGE (MT cells, mutant) and were then propagated in medium containing G418 (500 ⁇ g/ml). Clarified cell extracts were immunoprecipitated with affinity purified anti-VCIP-c-cyto rabbit polyclonal antibodies and analyzed by anti-HA immunoblotting.
• Figures 6B-D Formation of cell aggregates.
• Figure 6H The percentage of apoptotic nuclei was determined by scoring at least 300 cells from five randomly selected microscopic fields. Data are expressed as the mean + SD from triplicate samples. *, P ⁇ 0.05.
• Figures 6I-K Representative photomicrographs of Hoechst-stained V cells ( Figure 61), WT cells ( Figure 6J) and MT cells ( Figure 6K). Apoptotic nuclei are indicated by anows.
• FIGS 7A-0 show the effects of VCIP on adhesion of cadherin-deficient SW480 cells to HUVECs monolayer.
• SW480-C, SW480-WT, and SW480-MT cells were non-enzymatically detached from dishes, washed, passed through a cell strainer, counted, and labeled with a fluorescent dye (red) prior to experiments.
• HUVECs were seeded onto 12-well dishes in complete media and allowed to form a 100% confluent monolayer on the day of assay. Monolayer HUVECs were washed once each with PBS and HCMF, and then preincubated in HCMF supplemented with Ca 2+ /Mg 2+ for 10 min at 37°C.
• SW480 cells (0.25 x 10 6 ) were finely resuspendend in 500 ⁇ l of HCMF buffer supplemented with Ca 2+ /Mg 2+ , layered onto the monolayer, and allowed to attach at 37°C for 1 hr. Unattached cells were removed by washing with PBS and adherent cells were fixed with 4 % paraformaldehyde. The number of cells adhered to the monolayer were determined using a phase-contrast microscope. Experiments were performed at least three times in triplicates, with three independent clones. At least five random microscopic fields were selected for counting attached SW480 cells at 100X magnification. The total number of SW480-WT cells attached to the monolayer during first three experiments was designated as 100% cell attachment.
• Figures 7G-N HUVECs layered with SW480-WT cells pre-incubated with 10 ⁇ M GRGDSP peptide ( Figures 7G-H), 20 ⁇ M GRGDSP peptide ( Figures 71- J), 20 ⁇ g/ml affinity purified anti-VCIP-RGD antibody
• Figures 7K-L 40 ⁇ g/ml affinity purified anti-VCIP-RGD antibody ( Figures 7M-N). All antibodies were dialyzed against sterile 1 X TBS, pH 7.4 for 24 hours at 4°C to remove fraces of sodium azide and possible contaminants prior to use.
• Figure 70 Histogram showing adhesion of SW480 cells in the presence or absence of indicated substances.
• Figures 8A-G show VCIP-induced regulation of various infracellular signaling pathways.
• Cells were cultured exactly as described in Figure 6.
• V, WT and MT cells were solubilized, and extracts analyzed by immunoblotting with the indicated antibodies.
• Phospho-specific immunoblots were sfripped and re-probed with the conesponding total antibodies to confirm that equal amounts of protein were loaded across the lanes as shown. All blots shown are representative of those obtained in at least three separate experiments.
• Figure 8A ⁇ l integrin immunoblot. Anowhead indicates immature form of the ⁇ l integrin subunit.
• Figure SB pl20ctn immunoblot.
• HEK293 cells express two isoforms of pl20ctn, isoform- 1 (pl20) and isoform-2 (pllO); pl lO is less abundant in HEK293 cells.
• Figure 8C Phospho-FAK and total FAK immunoblots.
• Figure 8D Phospho-Akt and total Akt immunoblots.
• Figure 8E Phospho-Jnk and total Jnk immunoblots.
• Figure 8F Phospho-Erkl/2 and total Erkl/2 immunoblots.
• Figure 8G Phos ⁇ ho-GSK3 ⁇ and total GSK3 ⁇ immunoblots.
• Figures 9A-F show VCIP mediates heterophilic cell-cell interactions. Magnification, 200X. Bar, 40 mm. *, P ⁇ 0.05. The images of cell aggregates appear out of focus.
• Figure 9A Representative photomicrograph of a mixture of WT and MT cells labeled with red (Dil) and green (DiO) fluorescent dyes at 0 h.
• Figure 9B Small and large cell-aggregates were visible after 6 h; green anowheads indicate the most productive cell aggregation (yellow).
• Figure 9C Representative large cell aggregates are shown after 12 h.
• Figure 9D Cells co-incubated with the GRGDSP peptide for 12 h.
• Figure 9E Cells co- incubated with anti-VCIP-RGD antibody (25 ⁇ g/ml) after 12 h. All experiments were performed at least three times in triplicates.
• Figure 9F Histogram showing that the anti- VCIP-RGD ( ⁇ -VCIP) antibody and GRGDSP (RGD-) peptides dose-dependently inhibited cell aggregation, whereas control antibodies ( ⁇ -IgG) and peptides GRADSP (RAD-) did not.
• Figures 10A-H shows recombinant expression of VCIP-RGD protein induces interactions with ⁇ v ⁇ 3 and ⁇ 5 ⁇ l integrins.
• Figure 10A Affinity purified GST- fusion proteins were resolved on SDS ⁇ PAGE and stained with Coomassie Blue.
• Figure 10C Dose-dependent binding of ⁇ v ⁇ 3 integrin with recombinant GST- VCIP-RGD.
• Figures 10D-F Representative photomicrographs of adhesion and spreading of endothelial cells replated onto dishes coated with either fibronectin ( Figure 9D), vitronectin
• Adhesion blocking assays Endothelial cells were replated onto dishes coated with increasing concenfrations of affinity purified fusion proteins (1, 5 and 10 nM). Attached cells were stained, washed and absorbances of eluted dyes measured. To determine the effects of anti-integrin antibodies, dishes were coated with 10 nM GST-VCIP-RGD. Endothelial cells were pre-incubated at 4°C with increasing concenfrations of anti- ⁇ 5 ⁇ l
• P1D6 anti- ⁇ v ⁇ 3 (LM609), anti- ⁇ 2 ⁇ l (MAB 1998) and anti- ⁇ 3 ⁇ l (P1B5) monoclonal antibodies (1, 5 and 10 ⁇ g/ml) for 30 min, then washed and replated onto coated dishes. Attached cells were stained, washed and absorbances of eluted dyes determined. Three independent experiments were performed with several replicate samples in each experiment. Data are expressed as mean + S.D.; diamond, P ⁇ 0.01; asterisk, P O.001.
• FIG. 10H Clarified extracts prepared from [ 35 S]Cys/Met-labeled HdMVEC were pre- adsorbed and incubated with GST-VCIP-RGD in the absence (-) or presence (+) of GRGDSP peptide (lanes 1 and 2). The presence of ⁇ v ⁇ 3 and ⁇ 5 ⁇ l integrins (lanes 5 and
• Figures 11A-G shows tyrosine phosphorylation of Fak, Cas, She, Paxillin and Erk2. Dishes were coated with either fibronectin (Fn, 10 ⁇ g/ml), vitronectin (Vn, 10 ⁇ g/ml) or affinity purified GST-VCIP-RGD or -RGE fusion proteins (10 and 20 nM).
• FIG. 11A-E Equal amounts of protein ( ⁇ 1.0 mg) from clarified extracts were subjected to immunoprecipitation (IP) followed by immunoblotting using the indicated antibodies.
• Figure 11F Equal amounts of protein from total lysates (35 ⁇ g/lane) were subjected to immunoblotting with the anti-phospho-Erkl/2 antibody.
• Figure 11G The same membrane was stripped and re-probed with an anti-Erkl/2 antibody, showing that equivalent amounts of proteins used across the lanes.
• Figures 12A-I shows co-expression of VCIP with VEGF, and ⁇ v ⁇ 3 integrin in the tumor vasculature.
• Paraffin-embedded skin melanoma Figures 12A-F
• angioma Figures 12 G-I
• tumor tissue sections (4 ⁇ m) were subjected to indirect double immunostaining. The sections were sequentially incubated with affinity purified anti- VCIP-RGD (30 ⁇ g/ml) ( Figures 12A, D and G), anti-VEGF ( Figure 12B), anti-vWF ( Figure 12E) or anti- ⁇ v ⁇ 3 integrin (Figure 12H) monoclonal antibodies.
• FIG. 12C, F and I represent merged images of Figures 12 A-B, Figures 12D-E, and Figures 12G- H, respectively. Co-expression and co-incidences are indicated in yellow. Magnification, 100X. L, lumen. Bar, 25 ⁇ m.
• Figures 13A-D show quiescent endothelium lacks VCIP expression.
• Normal skin tissue section (4 ⁇ m) was processed as described above.
• Bright light photomicrograph of a skin section shows the architecture of normal tissue ( Figures 13 A).
• Sections were sequentially incubated with anti-VCIP-RGD (30 ⁇ g/ml) ( Figure 13B) and anti-CD31 /PECAM- 1 antibodies (20 ⁇ g/ml) ( Figures 13C). Sections were then washed and incubated with goat anti-rabbit IgG conjugated to Texas red (red) and goat anti-mouse IgG conjugated to FITC (green). Images were taken below saturation level with a Zeiss Axiovert-125 epifluorescent microscope equipped with a camera. Figure 13D represents the merged images of B and C. Images shown are representative of those obtained in at least three separate experiments. Magnification 100X. L, lumen. Bar, 25 ⁇ m.
• Figures 14A-H show schematic diagrams of various pLNCX2 retroviral constructs used in Figures 14-19. The relative positions of RGD, RGE, and RAD within the consfructs are indicated. Anows indicate the direction of transcription (5'-3'). Asterisk indicates the location of lipid phosphatase-dead motif.
• Figure 14P shows expression analysis of PAP2b/VCIP constructs in HUVECs. Whole cell extracts were analyzed by immunoprecipitation and western immunoblotting with anti-HA monoclonal antibodies (mAb). Arrowhead indicates IgG-heavy chain. Epitope tags: HA, hemaglutinin; FLAG, Flag-C2.
• FIGS 15A-K show retroviral infection of HUVECs with VCIP-RGD impedes their ability to migrate and close an artificial wound.
• HUVECs (70% confluent) in complete media were transiently infected with confrol (V), wild-type-PAP2b-RGD (WT), or mutant-PAP2b-RGD (MT) retroviral particles. After 24 hours post-infection, cells were washed once with PBS. Aliquots of cells were solubilized, expression of PAP2b/VCIP was analyzed by immunoprecipitation and western immunoblotting with indicated antibodies (Figure 15 A). The cells were analyzed for their ability to proliferate (Figure 15B).
• Figures 16A-K show PAP2b interacts with pl20 catenin.
• HUVECs were infected with pLNCX2-PAP2b-RGD (WT) retroviral particles and allowed to recover for 36 hours in complete media before stimulated with VEGF 165 for 6 hours.
• Cells were solubilized in modified RIPA, clarified lysates were immunoprecipitated with indicated antibodies and analyzed by western immunoblotting.
• Figure 16A Immunoblotting with anti-pl20 catenin (1.0 ⁇ g/ml).
• Figure 16B Immunoblotting with anti-HA (0.5 ⁇ g/ml).
• Figure 16C Immunoblotting with anti- VE-cadherin (lane 1), anti- ⁇ -catenin (lane 2), anti- ⁇ -catenin (lane 3).
• Figures 16D-K SW480 stably expressing wild-type PAP2b/VCIP (construct B) was solubilized in modified RIPA buffer and immunodepleted with anti-pan- cadherin monoclonal antibodies for 2 hours at 4°C. Lysate was recovered and absorbed with sepharose protein-G beads for 1 hour at 4°C to eliminate left-over (anti-pan-cadherin) IgG.
• Immunodepleted samples were immunoprecipitated with indicated antibodies and analyzed as shown: immunoblotting with anti-pl20 catenin (Figure 16D), anti- ⁇ -catenin (Figure 16E), anti- ⁇ -catenin (1.5 ⁇ g/ml) monoclonal ( Figure 16F), anti-PAP2b-cyto (0.5 ⁇ g/ml) polyclonal antibodies ( Figures 16G, H, I). Arrows indicate major isoforms of p 120 catenin polypeptides. Arrowheads indicate PAP2b polypeptides ( Figures 16G, H, I). All blots shown are representative of those obtained in at least three separate experiments.
• Figures 17A-H show interaction of PAP2b/VCIP with pl20 catenin in endothelial cells.
• Figures 17A-B Clarified cell lysates were prepared from endothelial cells infected with indicated PAP2b retroviral constructs A-E as shown in Figure 14 and subjected to immunoprecipitation with: anti-HA (lanes 1, 2, 4, 5 and 6) or anti-pl20 catenin (lane 3) and analyzed by immunoblotting with the indicated antibodies.
• Figures 17C-H Far western analysis. Endothelial cells were solubilized in RIPA buffer, and immunoprecipitated using indicated antibodies (5 ⁇ g/1.5 mg protein).
• FIG. 17C The membrane shown in Figure 17C was incubated with 2 ⁇ g/ml of Gst-PAP2b-cyto fusion protein for 1 hour, washed with 1XTBS + tween (0.1%) and analyzed by immunoblotting with anti-Gst monoclonal antibodies (2 ⁇ g/ml). Individual lanes from the blot was excised and probed with: anti- VE-cadherin (Figure 17D), anti-pl20ctn (Figure 17E), anti- ⁇ -catenin (Figure 17F), anti- ⁇ -catenin (Figure 17G), and ⁇ l -integrin (Figure 17H) monoclonal antibodies.
• Figure 17D anti- VE-cadherin
• Figure 17E anti-pl20ctn
• Figure 17F anti- ⁇ -catenin
• Figure 17G anti- ⁇ -catenin
• Figure 17H ⁇ l -integrin
• Figures 18A-K show regulation of pl20ctn and ⁇ -catenin by PAP2b/VCIP. Data shown are representative of those obtained in at least three separate experiments. Magnification, 100X (SW480), 200X (HUVECs).
• Figures 18A-B SW480 E-cadherin-deficient cells do not express PAP2b as illustrated by negative staining ( Figure 18 A) while pl20-_-catenin (pl20ctn) is diffusedly distributed in the cytoplasm ( Figure 18B) and ⁇ -catenin is mostly nuclear (Figure 18C).
• Figures 18D-I SW480 cells stably expressing WTPAP2b-RGD induce formation of cell-cell junction like structures.
• Figures 18D, E and G, H were sequentially incubated with indicated antibodies, followed by secondary donkey IgGs conjugated either to FITC (green) or Texas-Red (red) ( Figures 18D, E and G, H).
• Figures 18F and I are merged images of Figures 18D, E and G, H respectively.
• Figures 18J-K HUVECs were infected with confrol construct pLNCX2 ( Figure 18J) or pLNCX2- PAP2b-RGD ( Figure 18K). Recruitment of pl20ctn was analyzed by immunostaining with anti-pl20ctn monoclonal antibody.
• FIG 19A shows LEF-1 transcription assay.
• SW480 cells stably expressing the indicated retroviral constructs A-F were transiently transfected with TOPFLASH (5 ⁇ g/10 6 cells) together with tracer amount of ⁇ -galactosidase plasmids (1 ⁇ g/10 6 cells) for 3 hours with SuperFectTM (Qiagen) and allowed to recover for 36 hours in complete media containing 5% fetal calf serum. The amount of proteins and ⁇ - galactosidase activity were determined. Protein concentrations were adjusted for equivalent ⁇ -gal activities. LEF-1 luciferase activities were measured by Luciferase Assay
• Figures 20A-O show the effects of PAP2b/VCIP expression on tumor growth in athymic nude mice.
• Female nude mice (3-4 weeks old) were injected subcutaneously with SW480 cells ( ⁇ 2 xlO 4 ) expressing indicated PAP2b constructs: vector alone control, PAP2b-RGD wild-type, PAP2b-RGE mutant, PAP2b-C-cyto and PAP2b-PD phosphatase dead mutant. After 30 days, the visible primary tumor outgrowth was photographed.
• Figure 20F shows no visible growth of SW480 confrol cells beyond 2 mm in diameter.
• Figures 20G-J show primary tumors beyond 2-3 mm in size.
• Figures 20K-O show close-up images of experimental tumor growth in the athymic nude mice.
• Figures 21A-E show detection of human ALU sequences in tissues from athymic nude mice injected subcutaneously with SW480 cells expressing indicated PAP2b constructs: vector alone confrol (Figure 21A), PAP2b-RGD wild-type (Figure 21B), PAP2b-RGE mutant (Figure 21C), PAP2b-C-cyto ( Figure 21D) and PAP2b-PD phosphatase dead mutant ( Figure 21E).
• Genomic DNA (10 ng) from different tissues of the tumor-bearing mice were analysed for the presence of human ALU sequence by PCR.
• An internal control of mouse-glyceraldehyde-3 -phosphate dehydrogenase (m-GAPDH) was included.
• FIG. 22 shows anti-VCIP antibody blocks angiogenesis in vitro.
• Antibodies were dialyzed in sterile dialysis buffer (25 mM Tris, 175 mM sodium chloride, 2 mM potassium chloride pH 7.4) overnight to remove traces of azide and impurities. Integrity of antibodies was determined by SDS-PAGE. Activation and capillary morphogenesis of endothelial cells were performed in a 3D type I collagen matrix as described previously (Humtsoe et al., 2003). To detemiine the effects of specific antibodies on pre-formed capillaries, the method of Bayless et al. was used.
• mAbs were added to the culture medium at a concentration of 20 ⁇ g/ml. Fresh mAbs were added every 12 hours for a total of 60 hours.
• 3D matrices were fixed at 24, 36, 48, 60, and 72 hours by aspirating the medium, washing with PBS, and then fixing with 4% glutaraldehyde in PBS, pH 7.4, overnight at 4°C. Matrices were then washed with distilled water and embedded in paraffin according to the manufacturer's instructions (Richard Allen Scientific). Serial sections (4 ⁇ m) were prepared, dehydrated, stained with acidified eosin, and destained with distilled water.
• Capillaries were counted and photographed using a Zeiss Axiovert 25C microscope at 100X magnification. Each capillary tubule was sunounded by least 2 to 5 endothelial cells. Capillary formation was defined as the induction of a mhiimum of 3 separate capillary events within a single field. At least 5 random fields were counted for each sample. Experiments were performed in duplicate, using triplicate wells in each case, and results were expressed as mean + SEM. * p ⁇ 0.05; p ⁇ 0.01.
• Figures 23A-J are representative photomicrographs showing effects of specific mAbs on pre-formed capillaries. Endothelial cells were cultured in 3D collagen matrices in the presence of VEGF 165 . Cultures were treated with mAbs at 24 hours and at various time points indicated, the cultures were fixed, sections prepared, stained with eosin, and the number of capillaries counted as described above. The upper panels ( Figures 23A-E) were treated with anti-MHC class II mAbs, whereas the lower panels ( Figures 23H-J) were treated with anti-a ⁇ 3 integrin mAbs. Bar, 50 ⁇ M.
• VCIP/PAP2b mRNA was identified as a 3.4 kb transcript, not as a 1.6 kb transcript as described previously (Kai et al., 1997). No 1.6 kb PAP2b transcript was detected in any ofthe northern blot analyses described below.
• the cell membrane fraction prepared from 293 T cells over-expressing PAP2b showed phosphatase activity against phosphatidic acid that was independent of Mg 2+ , insensitive to N-ethylmaleimide exposure, and blocked by propranolol and sphingosine.
• VEGF, bFGF and PMA are able to induce expression of VCIP in three dimensional as well as monolayer cells.
• Cell surface biotinylation and FACS data indicated that VCIP is located on the cell surface.
• Refroviral-mediated elevated expression of wild-type VCIP in primary endothelial cells impeded cell migration and wound healing without altering proliferative potential of these cells.
• This observation suggested that VCIP might form a molecular complex on endothelial cells.
• a recent study showed cell-cell and basolateral sorting of VCIP (hLLP3) protein on polarized MDCK cells, while PAP2a (hLPPl) protein sorted on the apical surface.
• PAP2a contains a dityrosine (Y109/Y110) basolateral targeting motif that was first characterized in LDL receptor.
• the apical sorting of PAP2a is driven by the FDKTRL amino acid sequence, a similar motif that also occurs in cysteic fibrosis protein.
• basolateral and cell-cell localization of VCIP serves as mechanisms to promote integrin ligation at the cell-cell junction.
• VCIP mediates cell-cell interactions and promotes phosphorylation of GSK3 ⁇ and cAKT protein kinases. Furthermore, the C-terminal of VCIP directly associates with pl20catenin, which is likely to affect Wnt signaling pathway. The removal of the C-terminal tail of VCIP abolishes interaction with pl20catenin. Increased expression of VCIP stabilizes ⁇ -catenin in the cytoplasm and inhibits transcriptional activities through LEF-1. Removal of C-terminal cytoplasmic segment of VCIP augments LEF-1 transcriptional activities. The role of VCIP in angiogenesis can be further elucidated by structure-functional studies.
• VCIP exhibits an RGD sequence, and it promotes heterophilic cell-cell interactions and signaling.
• RGD motif of VCIP is a potent ligand for a subset of integrins.
• VCIP appears to be preferentially expressed in inflamed/angiogenic tissues.
• VCIP promotes 'heterophilic interactions', in that it can mediate both "homotypic” (like) and “heterotypic” (unlike) cell adhesions.
• VCIP-RGD could bind monocytes, and thereby enhance the adherence of neutrophils to endothelial cell monolayers.
• ⁇ l and ⁇ 2 integrins are known to mediate adherence of monocytes to endothelial and epithelial cells, an early event in the acute inflammatory response. It is also possible that activated endothelial cells could recruit carcinoma cells that express VCIP.
• carcinoma cells such as A431-like cells may utilize VCIP-RGD to recruit activated endothelial cells.
• Platelet integrin ⁇ llb ⁇ 3 may also interact with VCIP-RGD and contribute to platelet adhesion and aggregation. Lateral cell-cell interactions may provide a mechanism to impede or stop further migration of cells, thereby sequestering a subset of integrins from the basolateral surface ofthe cells towards cell-cell junctions. While interactions of endothelial cells with mesenchymal or smooth muscle cells may serve as a mechanism to promote recruitment of mural cells or pericytes, this may also promote maturation of blood vessels.
• the present invention identifies novel functions of PAP2b/VCIP. Since synthetic peptide and fusion proteins modeled after the second extracellular loop of VCIP bind selectively to ⁇ v ⁇ 3 and ⁇ 5 ⁇ l integrins, VCIP-derived peptides or proteins should inhibit specific cell-cell interactions. Such inhibitors of cell- cell interactions could be useful for developing novel therapeutic approaches to treat diseases where these interactions have clear pathological consequences, such as inflammation, thrombosis, atherosclerosis, restenosis and tumor-induced angiogenesis. Experiments can be designed to identify other molecules that may directly or indirectly function with VCIP, and examine how these factors may influence cell-cell interactions.
• the present invention provides methods of enhancing or inhibiting cell-cell interaction by modulating the expression and function of VCIP (SEQ ID NO: 13).
• VCIP SEQ ID NO: 13
• such cell-cell interaction contributes to a biological process such as normal cell cycle progression, unwanted cell cycle progression, vascular malformation, expansion of atherosclerotic lesions, invasion of tumor cells, inflammation, cell motility, or angiogenesis.
• the cell-cell interaction is mediated by integrins.
• VCIP expression can be enhanced by over-expressing VCIP in a cell, resulting in enhanced cell- cell interaction.
• a gene encoding VCIP can be delivered to a cell by methods generally known in the art.
• gene delivery can be accomplished by a viral vector such as adenoviral vector or by a non-viral gene delivery system such as high pressure gene delivery system ("Genegun”) or liposome.
• a viral vector such as adenoviral vector
• a non-viral gene delivery system such as high pressure gene delivery system ("Genegun") or liposome.
• Over-expression of VCIP may promote cell- cell adhesion junction formation in patients with compromised blood-brain barrier functions. This therapeutic approach may stop edema and hemorrhage following traumatic brain injury such as gun shot wound.
• Over-expression of VCIP may also enhance angiogenesis in patients who need growth of new blood vessels for treating various ischemic diseases.
• the function of VCIP and cell-cell interactions can be inhibited by blocking the binding of integrin to cell surface VCIP. Binding of VCIP to its ligand can be blocked by antibodies directed against a VCIP peptide comprising a RGD sequence or by a RGD-containing peptide derived from VCIP. The function of VCIP can also be blocked by anti-sense VCIP oligonucleotides. These methods of blocking and inhibiting the binding of VCIP are useful in treating an individual having a pathological condition resulted from undesirable integrin-mediated cell-cell interaction. In general, such pathological condition includes, but is not limited to, stroke, thrombosis, tumor growth, metastasis, arthritis, cardiac infarction, psoriasis, diabetic retinopathy, inflammation, and angiogenesis.
• the present invention provides a fragment of VCIP that contains a RGD sequence.
• Such peptide is useful in inhibiting the binding of VCIP to its ligand.
• Representative examples of such peptides include, but is not limited to, SEQ ID NO: 20 and 32.
• the present invention further provides vectors encoding such peptides as well as antibodies directed against such peptides. These antibodies can be incorporated into a kit useful for detecting VCIP in an individual having a disease such as pathological angiogenesis, inflammation, arthritis, psoriasis, atherosclerosis, or metastatic disease.
• HBVECs Human umbilical vein endothelial cells
• HdMVECs human dermal microvascular endothelial cells
• CASMCs carotid artery smooth muscle cells
• AoSMC aortic smooth muscle cells
• ECM molecules endotoxin-free fetal bovine serum, antibiotics, heparin, 100X ITS (insulin, fransfenin and selenium), M199 media, anti- ⁇ 5 ⁇ l (P1D6) and anti- ⁇ 3 ⁇ l (P1B5) antibodies and Superscript II reverse transcriptase enzyme were obtained from Invitrogen.
• bFGF Basic Fibroblast growth factor
• hrVEGF 165 human recombinant vascular endothelial growth factor
• Bovine skin-derived type I collagen (3.0 mg/ml) solution was purchased from Cohesion Inc.
• Multiple tissue northern blot, cDNA amplification kit and human placental cDNA library in _Triple-Ex vector were purchased from Clontech Laboratories, Inc.
• Anti-phosphospecific antibodies were purchased from New England Biolabs.
• Hybridomas producing the anti-human ⁇ l ⁇ l integrin antibody (clone TS2/7) were obtained from ATCC.
• Anti- ⁇ 2 ⁇ l (MAB 1998), anti-avb3 (LM609) and VE-cadherin (MAB 1989) antibodies were procured from Chemicon.
• Mouse anti-pl20catenin (clone 15D2) monoclonal antibody was obtained from
• Synthetic peptides LSPVDIIDRNNHHNM SEQ ID NO:l
• EGYIQNYRCRGDDSKVQEAR SEQ ID NO:2
• Three- dimensional matrix gel was prepared by gently mixing a cold solution of bovine skin- derived type I collagen solution (2.1 mg/ml) with media Ml 99, IX ITS, hrVEGF 165 (100 ⁇ g/ml) and glutamine (2.4 mM). The pH was adjusted to 7.5 with 0.1 N sodium hydroxide and sterile water was used to adjust the final volume. Proliferating endothelial cells in the third or fourth passage were cultured in complete media and gently resuspended in complete Ml 99 media at a concentration of 4X10 5 cells/ml.
• tubulogenic media including Ml 99 media, IX ITS, 20% adult human serum-AB and hrVEGF 165 (100 ng/ml).
• tubulogenic media is used to describe the media that induces formation of 'capillary (or tubule) morphogenesis' of endothelial cells grown in 3D gels.
• endothelial cells embedded in three-dimensional type I collagen in the presence of 20% human adult serum- AB ⁇ 100 ng ml hrVEGF 165 supplied every 6 h) were fractionated on an agarose gel containing formaldehyde.
• the following primers were used: VCIP-forward 5'-
• modified RIPA buffer 50 mM HEPES pH 7.5, 1.0% Triton X-100, 0.1% SDS, 0.25% deoxycholate, 150 mM sodium chloride, 1 mM EDTA, 25 mM sodium fluoride, 1 mM sodium pyrophosphate, 2 mM sodium orthovanadate and appropriate concenfrations of various protease inhibitors).
• HEK293 cells (5 x 10 6 ) were transfected with either pEGFP-C3, pEGFP-N3, pEGFP-C3-VCIP or pEGFP-N3-VCIP using Superfect-LiposomeTM (Qiagen). Biotinylation of cell surface proteins was carried out according to published procedures (Gottardi et al., 1995). Immunoprecipitation, immunoblotting and immunodetection protocols were all performed as described previously (Mainiero et al, 1995, 1997; Wary et al., 1996, 1998, 1999a,b). EXAMPLE 5
• HdMVECs Human dermal microvascular endothelial cells (3 x 10 7 ) were deprived of growth factors in Cys/Met-free DMEM for 8 h. The cells were then incubated with 3 mCi of [ 35 S]Cys/Met (specific activity 1170.0 Ci/mmol) for 3 h at 37°C in Cys/Met-free media in the presence of lx ITS. After 3 h, the cells were rinsed twice with complete media and allowed to recover in complete media for 1 h at 37°C.
• the cells were then washed and solubilized in 4 ml of complete cell extraction buffer (CCEB: 50 mM HEPES pH 7.4, 150 mM sodium chloride, 1% Triton X-100, 0.1% ⁇ -octylglucoside, 1 mM MgCl 2 , 2 mM CaCl 2 , with freshly added 2 mM PMSF, 10 ⁇ g/ml aprotinin, 5 ⁇ g/ml leupeptin and 10 ⁇ g/ml pepstatin-A as protease inhibitors).
• complete cell extraction buffer CCEB: 50 mM HEPES pH 7.4, 150 mM sodium chloride, 1% Triton X-100, 0.1% ⁇ -octylglucoside, 1 mM MgCl 2 , 2 mM CaCl 2 , with freshly added 2 mM PMSF, 10 ⁇ g/ml aprotinin, 5 ⁇ g/ml leupeptin
• Cell extracts were clarified, pre-adsorbed once with 1.5 ml of packed Sepharose beads coupled to GST-fusion proteins (2 mg/ml) and once with 1.0 ml (packed) anti-mouse IgG agarose for 2 h each at 4°C.
• Pre-adsorbed lysates were divided into two tubes, and 7 ⁇ g of GST-VCIP- RGD fusion protein was added to each sample.
• One of the tubes included 25 ⁇ M of the synthetic soluble peptide GRGDSP (SEQ ID NO: 11) which is known to disrupt ⁇ 5 ⁇ l integrin-fibronectin interaction.
• GST-pull down was carried out at 4°C for 8 h, complexes washed once with CCEB, three times with GST-fusion protein wash buffer (50 mM HEPES pH 7.4, 150 mM sodium fluoride, 5% glycerol, 0.5% NP-40, 1 mM CaCl 2 and 1 mM MgCl 2 ) and one final wash with lx TBS pH 7.4.
• the contents of the other tube were resuspended in 0.5 ml of dissociation buffer (10 mM Tris pH 7.4, 0.75% SDS, 1% Triton X-100 and 250 mM NaCl), boiled for 10 min, centrifuged immediately and the beads were discarded. The supernatant was diluted with 4 ml of dilution buffer: 10 mM Tris pH 7.4, 100 mM NaCl, 1.0% Triton X- 100, 2 mM CaCl 2 and 2 mM MgCl 2 .
• PCR primers containing BamHI (5') and Hindlll (3') restriction sites were designed.
• the GFP gene was inserted in-frame with the VCIP gene (on either the N-terminus or C-terminus) into the mammalian expression plasmids pEGFP-N3 or pEGFP-C3, thereby producing pEGFP-VCIP-N3 or pEGFP-VCIP-C3 fusion proteins ( Figure 3A-D).
• human VCIP cDNA was subcloned into pLNCX2 (Clontech) immediately downstream of the human CMV immediate early promoter.
• Two-step PCR was used to insert three copies of an HA-tag (YPYDVPDYA, SEQ ID NO: 12) at the N-terminus of the VCIP cDNA and to mutate the wild-type RGD sequence in one of the proteins to RGE ( Figure 3E-G).
• the two-step PCR method has been described previously (Wary et al., 1996).
• Amphopack- 293 packaging cells (Clontech) were transfected with pLNCX2 (V), pLNCX2- VCIP-RGD (WT) and pLNCX2-VCIP-RGE (MT) using SuperfectTM liposome (Qiagen).
• pLNCX2-VCIP-RGD-HEK (WT) and pLNCX2-VCIP-RGD-HEK (MT) cells were detached from dishes with 0.025% trypsin and 2 mMEDTA, washed with PBS and passed through a cell strainer.
• HCMF buffer (20 mM HEPES pH 7.4, 137.5 mM NaCl, 5.0 mM KCl, 0.35 mM Na 2 HPO 4 .7 H2O, 4.5 mM glucose and 10 mM CaCl 2 ) supplemented with 5 mM Ca 2+ , 1 mM Mg 2+ , 10 ⁇ g/ml of
• apoptosis assay cells were deprived of growth factors for 24 h, then incubated in defined medium for 28 h. Attached and unattached cells were combined, fixed with cold 20 mM glycine-HCl pH 2.0 and stained in suspension with Hoechst 33258 dye (0.5 ⁇ g/ml). Cells were examined under a Zeiss Axiovert-125 fluoroscope. The presence of more than two visible nuclear fragments was considered as a single apoptotic event. Apoptotic events were counted from at least five random microscopic fields.
• Solid phase ligand binding assays were performed according to a previously published procedure (Orlando and Cheresh, 1991). Briefly, soluble ⁇ 2 ⁇ l, ⁇ 5 ⁇ l, ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins (1 ⁇ g/ml in a solution containing 20mM Tris pH 7.4, 150mM NaCl, 1 mM CaCl 2 , ImM MgCl 2 and 1 mM MnCl 2 ) were immobilized onto 96-well microtiter plates at 4°C. Wells were washed and blocked with 0.5% BSA.
• GST, GST-VCIP-RGD and GST-VCIP-RGE ligands were added (50-350 ng per well in a solution of TBS pH 7.4) and incubated at 37°C for 1 h. After washing, the wells were incubated with the anti-GST (sc-138, Santa Cruz) monoclonal antibody for 1 h, followed by washing and incubation with a horseradish peroxidase (HRP)-conjugated mouse secondary antibody. Plates were then washed again and the ABTS substrate (Bio-Rad) was added. All washing steps were carried out using PBS. Absorbances were read at 405 nm, and non-specific binding values were adjusted against BSA.
• HRP horseradish peroxidase
• endothelial cells were detached, washed with PBS and resuspended in Ml 99 media containing 1 mM CaCl 2 and 1 mM MgCl 2 in the absence of serum or growth factors.
• Endothelial cells (2 x 10 5 cells) were replated onto 24- well tissue culture plates coated with 1, 5 or 10 nM affinity purified GST-VCIP-RGE or GST-VCIP-RGD fusion proteins. Cells were allowed to reattach for 45 min, then washed, fixed with 4% paraformaldehyde, stained with 0.5% crystal violet for 5 min and then washed extensively with water. Absorbances were measured at 540 nm.
• Double mmix ostaining of paraffin-embedded tumor sections (4 ⁇ m) was performed following antigen retrieval. Specimens were subjected to microwave treatment (1000 W) in citrate buffer pH 6.0, four times for 5 min each. Peroxidase activity was then inhibited by the addition of 3% H 2 0 2 in PBS for 20 min, followed by blocking with 3% BSA in PBS. Sections were then incubated with the affinity purified anti-VCIP-RGD antibody (20 ⁇ g/ml), followed by either anti- VEGF (30 ⁇ g/ml), vWF (50 ⁇ g/ml) or anti- ⁇ v ⁇ 3 integrin (30 ⁇ g/ml) antibodies.
• VCIP VEGF and type I collagen inducible protein
• VCIP mRNA was most strongly expressed in human heart and placenta, tissues that are highly vascularized ( Figure IE).
• Figure 1G expression of VCIP in monolayer endothelial cells treated with bFGF, VEGF and PMA was examined ( Figure 1G). It was found that all three cytokines were equally able to induce expression of VCIP. This pattern of regulation was identical to that ofthe human receptor for urokinase plasminogen activator (uPAR) expression (Figure IH). ⁇ -actin and GAPDH were mcluded as controls and were not regulated under any of these conditions ( Figure II and J).
• uPAR urokinase plasminogen activator
• the VCIP gene was cloned to investigate its possible role in capillary morphogenesis of endothelial cells.
• the investigators sequenced several 3' ends of RT-PCR products derived from a pool of 3' and 5' RACE products using DNA sequence information from clone 33 A, as shown in Figure IK. Sequencing of a -2.0 kb 3' region of a putative gene did not exhibit an open reading frame (ORF). Two 5' probes were generated from this 2.0 kb fragment, and used to screen a human placental cDNA library, resulting in the recovery of six _-phage cDNA clones (Figure IK).
• the complete cDNA sequence of VCIP has been deposited under the accession No. AF480883, and the deduced amino acid sequence of VCIP is shown in Figure IL.
• Database searches with the VCIP coding sequence and analysis of the deduced amino acid sequence revealed that VCIP has a consensus lipid phosphatase motif and an RGD cell attachment sequence in the second extracellular domain, whereas the cytoplasmic domains of VCIP lack any known enzymatic features or motifs.
• the lipid phosphatase motif of VCIP is shown in comparison with other known lipid phosphatase motifs in Figure IM.
• VCIP vascular endothelial cells
• HdMVECs Human dermal microvascular endothelial cells
• VCIP was expressed most strongly in cells that were stimulated with VEGF ( Figure 3A, lane 2).
• VEGF 165 Neither PMA nor VEGF 165 induced expression of VCIP in carotid artery smooth muscle cells (CASMCs), whereas VEGF 165 increased VCIP levels in aortic smooth muscle cells (AoSMC), but PMA did not. VCIP levels were strongly induced by PMA and EGF in epidermoid carcinoma (A431) cells, whereas TNF- ⁇ and bFGF had no effect in this cell type.
• Transfected cells were detached from culture dishes and subjected to cell surface biotinylation. Proteins were subjected to immunoblotting or immunoprecipitation with anti-GFP antibodies.
• Cells transfected with the control GFP vector exhibited a -30 kDa GFP-immunoreactive band ( Figure 5A, lanes 1 and 2), whereas a GFP- immunoreactive band of ⁇ 68 kDa was detected in lysates from cells transfected with the GFP- VCIP expression vector ( Figure 5B, lanes 3 and 4).
• Retroviral Transduction of VCIP Promotes Cell-Cell Interactions
• HEK293 cells stably expressing wild-type VCIP pLNCX2-VCIP-RGD- HEK, WT
• mutant VCIP pLNCX2-VCIP-RGE-HEK, MT
• vector alone pLNCX2- HEK, V
• pLNCX2-HEK (V) was chosen as a control because it is has no known effect on cells.
• WT cells were also incubated with several peptides modeled after the VCIP-RGD region.
• WT cells were cultured in the continuous presence of an anti- VCIP-RGD antibody (25-50 ⁇ g/ml) and NYRCRGDDSK (SEQ ID NO:20) (10-50 nM)
• the size, the speed of formation and the number of such cell aggregates were reduced (Table 1).
• no reduction in cell aggregation was observed in cells incubated with the mutant peptides NYRCRADDSK (SEQ ID NO:21) (10-50 nM) or NYRCRGEDSK (SEQ ID NO:22) (10-50 nM).
• Incubation with the antibody or peptides did not induce toxicity or cell death.
• the cell aggregation observed in WT cells was specific, in that cells transfected with pLNCX2-HEK or pLNCX2-VCIP-RGE-HEK did not exhibit such phenotype ( Figure 6B and D).
• VCIP-RGD construct attached to monolayer HUVECs, whereas cells expressing pLNCX2 or pLNCX2-VCIP-RGE did not.
• Adhesion of pLNCX2-VCIP-RGDSW480 cells to monolayer HUVECs was blocked by incubation with the anti-VCIP-RGD antibody and the GRGDSP (SEQ ID NO: 11) peptide in a dose-dependent manner ( Figure 7), whereas confrol substances had no significant effect on adhesion.
• HEK cells stably expressing Substance Concentration i % cell aggregates ⁇ LNCX2 (vector alone) (V) 0 pLNCX2-VCIP-RGD (WT) 94 ⁇ 5.5 a pLNCX2-VCIP-RGE (MT) 0 pLNCX2-VCIP-RGD (WT) Anti-rabbit IgG 25 ⁇ g/ml 90 ⁇ 7.3 pLNCX2-VCIP-RGD (WT) Anti-rabbit IgG 50 ⁇ g/ml 91 ⁇ 8.8 pLNCX2-VCIP-RGD (WT) Anti-VCIP-RGD 25 ⁇ g/ml 73 ⁇ 7.4 pLNCX2-VCIP-RGD (WT) Anti-VCIP-RGD 50 ⁇ g/ml 36 ⁇ 12.5 a pLNCX2-VCIP-RGD (WT) NYRCRGDDSK 10 nM 67 ⁇ 15.7 pLNCX2-VCIP-
• ⁇ l integrin and pl20catenin (pl20ctn) protein levels were measured.
• the phosphorylation state and total protein levels of Fak, Akt, GSK3 ⁇ and Erk2 protein kinases, which play roles in adhesion-mediated cell proliferation, survival and migration were also measured. Enzymatic activation of these protein kinases is accompanied by an increase in phosphorylation state, ⁇ l integrin immunoreactivity levels were similar in V, WT and MT cells ( Figure 8A).
• pl20ctn immunoreactivity levels were increased in WT cells, as compared with V or MT cells.
• the phosphorylation state of Fak, Akt and GSK3 ⁇ were increased in WT cells, as compared with
• VCIP-RGD could act as a cell- associated integrin ligand. Thereby, VCIP-RGD could promote 'cell ⁇ cell interactions' by specifically recognizing ⁇ v ⁇ 3 and ⁇ 5 ⁇ l integrins presented on adjacent cells.
• 293HEK cells express high level of ⁇ 5 ⁇ l, but somewhat relatively low in ⁇ v ⁇ 3 integrin heterodimer
• WT cells were mixed with cells expressing high levels of the ⁇ 3 integrin subunit to evaluate the effects on cell aggregation.
• 293HEK cells were stably transfected with the wild-type human ⁇ 3 integrin subunit. Expression levels were determined by FACS and western analyses. Mixing of WT cells with ⁇ 3 integrin-293HEK cells quickly resulted in significant cell aggregation within 3-6 h (data not shown).
• Wild-type glutathione S-transferase (GST)-VCIP-RGD and mutant GST- VCIP-RGE fusion proteins were affinity purified and visualized by Coomassie Blue staining on SDS-PAGE as shown in Figure 10A. Both the wild-type and mutant proteins migrated at the predicted size of 34 kDa.
• FIG. 10D-F Representative photomicrographs of optimal adhesion and spreading of endothelial cells plated for 45 min on fibronectin, vitronectin and GST-VCIP-RGD are shown in Figure 10D-F. Adhesion of endothelial cells to GST-VCIP- RGD was comparable to that observed in wells coated with vitronectin and fibronectin substrates.
• ⁇ v ⁇ l, ⁇ v ⁇ 5 and ⁇ v ⁇ 6 integrins may also mediate the interaction between endothelial cells and VCIP-RGD to some extent.
• adhesion of endothelial cells to VCIP required the presence of Ca 2+ and Mg 2+ , as the addition of 2.5 mM EDTA (pH 7.4) for 5 min caused the cells to detach from tissue culture dishes.
• VCIP-RGD acetylated NYRCRGDDSKVQE
• attached cells i.e. cells that were plated on VCIP-RGD-coated wells
• clarified cell lysates were obtained from [ 35 S]Met/Cys labeled HUVECs and subjected to affinity chromatography. Lysates were pre-adsorbed twice with GST-Sepharose beads to remove proteins that interact with GST-Sepharose beads non-specifically. Pre-adsorbed lysates were incubated with GST- VCIP-RGD fusion proteins (10 ⁇ g per 3 mg lysate) in the presence or absence of 25 ⁇ M
• GRGDSP (SEQ ID NO:l 1). The beads were then extensively washed. To determine whether integrins were present in the GST-VCIP-RGD pull-down complex, the contents of a tube that did not receive the GRGDSP (SEQ ID NO: 11) peptide was boiled in a dissociation buffer containing 0.5% SDS. The samples were equally divided into three tubes and diluted with cold immunoprecipitation dilution buffer to adjust the concenfration of SDS to ⁇ 0.1%. The samples were then immediately subjected to immunoprecipitation with indicated antibodies (Figure 10H). The results showed that the GST-VCIP-RGD pull-down complex indeed contained anti- ⁇ v ⁇ 3 and - ⁇ 5 ⁇ l immtmoreactiviti.es ( Figure 10H, lanes 4 and 5).
• Adhesion of Endothelial Cells Through VCIP-RGD Induces Integrin-Mediated Signaling Adhesion of cells to extracellular matrix proteins promotes clustering of integrins at the plane of the plasma membrane. In addition to promoting structural support, this event nucleates formation of a complex of signaling-competent intracellular proteins.
• pl25FAK, p46/52Shc, pl30Cas and paxillin were immunoprecipitated and subjected to immunoblotting with various phospho-specific antibodies.
• Serum- and growth factor-starved HUVECs were allowed to attach and spread on dishes coated with optimal concentrations of fibronectin, vitronectin, GST-RGD-VCIP and GST-VCIP-RGE.
• Cells were harvested after 30 and 60 min at 37°C. Cells were then solubilized, clarified, pre-adsorbed, immunoprecipitated and subjected to immunoblotting with various antibodies as shown in Figure 11.
• VCIP-RGD induced tyrosine phosphorylation of Fak, Cas, She and paxillin at both 30 and 60 min.
• the signal intensities induced by expression of GST-VCIP-RGD were comparable to those induced by fibronectin and vitronectin.
• GST-VCIP-RGE did not induce detectable tyrosine phosphorylation of Fak, Cas, She or paxillin.
• Cells that were replated onto GST-VCIP-RGE appeared rounded. Sfripping and reprobing blots with anti-pl25FAK, anti-pl30Cas, anti-She and anti-paxillin antibodies showed that equal amounts of these proteins were present under all experimental conditions (data not shown).
• Angiogenesis is required for the growth and survival of all solid tumors.
• tumor sections were immunostained with an anti-VCIP-RGD antibody.
• the specificity of affinity purified anti-VCIP-RGD was confirmed by ELISA, western immunoblotting and immunolabeling experiments.
• Anti-VCIP-RGD reacted specifically with the GST-VCIP-RGD fusion protein, but did not react with GST-VCIP-RGE or GST alone.
• the anti-VCIP-RGD antibody did not react with other RGD-containing extracellular matrix molecules such as fibronectin, vitronectin, or type I collagen.
• Tissue sections were initially examined by mimunostaining with anti-platelet endothelial cell adhesion molecule- 1 (PECAM- 1, also known as CD31), anti-VE (vascular endothelial)-cadherin and anti-von Willebrand Factor (vWF) antibodies to establish the presence of endothelium.
• PECAM- 1 anti-platelet endothelial cell adhesion molecule- 1
• VE vascular endothelial
• vWF anti-von Willebrand Factor
• Enriched expressions of VEGF and ⁇ v ⁇ 3 integrin are common in angiogenic tissues, and are associated with invasion and growth of solid tumors. An increase in the levels of vWF expression is considered to be a negative prognostic factor for tumor-induced angiogenesis.
• EXAMPLE 21 Overexpression of PAP2b/VCIP Impedes Endothelial Cell Migration Activated endothelial cells display a highly motile phenotype. This motile behavior of endothelial cells is largely mediated by integrins, and it is considered to be a crucial event for angiogenesis. Sprouting of new blood vessels requires cell division in preformed endothelial tissues, such as the wall of a blood vessel, and this proliferation is accompanied by robust endothelial cells migration. Regulatory mechanisms must exist to counter migratory activity of endothelial cells, so that unnecessary (or unwanted) angiogenesis can be prevented. In view ofthe above results that showed VCIP-RGD serves as a cell associated integrin hgand, the present example evaluates the effect of elevated expression of VCIP on endothelial cells motility.
• Retroviral vector pLNCX2
• amphotropic packaging cell line 293HEK
• Preparation of recombinant cDNA constructs for pLNCX2-PAP2b-WT and -MT has been described previously (Humtsoe et al., 2003). Additional constructs were generated using the existing pLNCX2- PAP2b-WT as template with restriction ends BamH I and Cla I.
• a phosphatase inactive or dead (PD) form of PAP2b was generated by double mutation of K148A and R155A by two- step PCR strategy.
• the primers used were, forward: 5'- GCCGGATCCATGCAAAACTACAAGTACGAC-3' (SEQ ID NO:24) and reverse: 5'- GAGGAGCCAGGCGCCCTATGGACACTGCGGCAAT-3' (SEQ ID NO:25); forward: 5'-TGCCGCAGTGTCCATAGGGCGCCTGGCTCCTCA-3' (SEQ ID NO:26) and reverse : 5 ' -GCGATCGATCTACATCATGTTGTG-3' (SEQ ID NO:27).
• HUVECs from passage 3-4 were used for the expression of confrol (V), wild- type PAP2b-RGD (WT), or mutant PAP2b-RGE (MT) by viral transduction.
• MT-PAP2b- RGE represents a single mutation at position D184E.
• construct A To monitor the non-toxicity of the viral particles used for infection, confrol supernatants generated from vector alone (construct A) were used. Expression levels were determined by immunoprecipitation and western immunoblot assay showing comparable levels of wild-type (construct B) and mutant (construct C) proteins ( Figure 15 A). As determined by Brdu inco ⁇ oration and monitored for the indicated time period, there was no defects observed in cellular growth between the control, MT or WT expressing cells ( Figure 15B).
• wound cells were incubated in a defined media and their ability to repopulate the wounded area was monitored for 0, 5, and 10 hours.
• About 40-60% confluent HUVECs on 12-well culture plates were infected with vector alone, PAP2b-WT or PAP2b-MT retroviral particles overnight.
• the cells were replenished with fresh media and allowed to grow to form confluent monolayer for about 12-24 hrs.
• Confluent monolayer cells were injured by sterile 200 ⁇ l micropipet tip, washed twice in sterile PBS, and allowed to recover in defined Ml 99 media. After specific times the plates were removed, washed in PBS and fixed in 4 % paraformaldehyde.
• Transwell cell migration assay was performed using VCIP infected cells. Migration assay was canied out using modified chemotactic Transwell Boyden (8.0 ⁇ M) chambers (Schor et al., 1996). Endothelial cells infected with retroviral constructs were detached non-enzymatically, washed once with complete media, followed with PBS, and resuspended in defined M199 media (M199 + 1 X ITS [insulin, fransfenin and selenium-A].
• Top chamber was filled with 500 ⁇ l media containing 2.5 X 10 4 cells and the lower chamber was filled with 500 ⁇ l of defined media. Following 6 hours at 37°C in CO 2 incubator, cells that remained on the upper chamber were gently removed by cotton Q-tips. Cells that migrated to the lower side of filter were fixed with 4 % paraformaldehyde and stained with 0.5% crystal violet. Cell number was counted using a phase confrast microscope. A minimum of 10 random fields at 100X magnification were selected for each chamber-filter. Experiments were performed tliree times with triplicates.
• This example examines the biochemical basis for the effects of PAP2b/VCIP on endothelial cell migration.
• Molecular organization of cell-cell contacts often requires participation of junction proteins and cytoskeletal elements.
• sequence analysis of the cytoplasmic domains of PAP2b/VCIP revealed no known protein binding motifs or enzymatic features. Therefore, PAP2b/VCIP interaction with known intracellular proteins that may be involved in cell-adherent junctional organization was examined. To do this, monolayer HUVECs infected with full length PAP2b (WT) and stimulated with VEGF 165 were solubilized in modified RIPA buffer, clarified, and subjected to coimmunoprecipitation analysis.
• pl20catenin which was initially identified as a V-Src substrate, has been implicated in cell-cell adhesion, signaling and tumor progression. Because pl20catenin is known to interact with cadherin when present in the cell-cell junction, the association of PAP2b/VCIP with pl20catenin was examined. For this purpose, E-cadherin deficient SW480 (human colon carcinoma) cells were made to stably express WT-PAP2b prior to pl20catenin interactions studies. Cell lysates prepared in RIPA buffer were clarified and immunoprecipitated with anti-HA, anti-pl20ctn, anti- ⁇ -catenin, anti- ⁇ -catenin and anti- ⁇ l monoclonal antibodies.
• anti-HA and anti-pl20ctn monoclonal antibodies co-precipitated pl20ctn and PAP2b/VCIP reciprocally ( Figure 16D). Similar to results using primary endothelial cells, anti-PAP2b immunoblots did not contain anti- ⁇ -catenin or anti- ⁇ -catenin immunoreactivities
• Anti-HA monoclonal antibody did not co-precipitate any detectable PAP2b or pl20ctn from cells infected with vector alone (construct A) ( Figures 17A-B, lane 1).
• Anti-HA mAb coprecipitated pl20ctn immunoreactivities from cells infected with constructs B, D, F but not E (constructs are shown hi Figure 14).
• Construct E lacks entire C-terminal cytoplasmic segment of VCIP.
• SW 80 cells stably expressing various PAP2b/VCIP constructs were used.
• PAP2b-WT to bind pl20catenin
• its ability to recruit pl20catenin at the pericellular cavity was evaluated.
• Endogenous PAP2b protein was undetectable in SW480 cells ( Figure 18A).
• endogenous pl20catenin remained evenly distributed in the cytoplasm ( Figure 18B), whereas ⁇ -catenin was exclusively localized in the nucleus ( Figure 18C).
• Expression of wild-type PAP2b resulted in recruitment of pl20catenin to the cell-cell contact sites ( Figures 18D-F).
• control wild-type VCIP (RGD), mutant VCIP (RGE), mutant VCIP-phosphatase dead and mutant VCIP-cyto constructs were maintained in DMEM containing 7.5 % FBS and 200 ⁇ g ml "1 geneticin (G418). Generation of constructs, infections and establishment of stable lines have been described above. The expression of PAP2b/VCIP protein was confirmed by Western blotting prior to injection to the mice.
• SW480 cells expressing VCIP- RGD WT
• SW480 cells expressing VCIP- RGD grew drammatically within 30 days with visibly distinct vasculature (angiogenic blood vessels) and localized hemonhage, suggesting aggressive nature of tumor growth.
• SW480 cells expressing VCIP- ⁇ -C-cyto showed robust tumor growth and neovasculature formation comparable to VCIP-RGD-WT.
• SW480 cells expressing phosphatase-inactive (phosphatase-dead) form of VCIP induced tumor growth and extensive angiogenesis.
• a ruler in centimeter scale was placed next to the tumor mass to show the extent of tumor growth ( Figures 20 F, G, H, I and J).
• Figures 20 K, L, M, N and O showed close-up images ofthe experimental tumor in nude mice.
• tumor growth and tumor angiogenesis can also be determined by metastatic foci formation at distant sites.
• brain, lung, liver, spleen, and kidney tissues were subjected to PCR to identify the presence of human tumor cells as determined by human Alu sequences.
• This assay is its sensitivity, i.e. as few as 50 tumor cells per 10 8 host cells can be detected.
• Tissues from sacrificed mice were snap-frozen in liquid-nitrogen and stored at — 80°C for later use.
• DNA was purified using Qiagen RNA/DNA mini kit according to manufacturer's instruction (Qiagen, Inc.). Purified DNAs were dissolved in deionized water, quantified and stored at -20°C until use.
• ALU-PCR strategy was employed.
• the oligonucleotides for human ALU were: sense, 5'-GTTGCCCAAGTTGGAGTGCAATGG-3' (SEQ ID NO:33) and antisense, 5'-ACAATGGCTCACGCCTGTAATCCC-3' (SEQ ID NO:34).
• Ten nanograms each of genomic DNA extracted from various mouse tissues were used in a final 25 ⁇ l reaction using Taq PCR master mix Kit (Qiagen, Inc).
• PCR parameters were set as initial denaturation 94°C, 10 min; denaturation 94°C, 1.5 min; annealing 55°C, 1.5 min; and extension 72°C, 2 min for 25 cycles followed by final extension of 72°C for 7 min.
• mouse glyceralaldehyde-3-phosphate dehydrogenase (GAPDH) was included using primers: sense, 5'-TGGAGTCTACTGGTGTCTTCACCACCATG-3' (SEQ ID NO:35) and antisense, 5'-GCAGGAGACAACCTGGTCCTCAGTG-3' (SEQ ID NO:36).
• Capillary morphogenesis of endothelial cells was performed in a 3D type I collagen matrix as described previously (Humtsoe et al., 2003) to evaluate the ability of anti- PAP2b-RGD (anti-VCIP-RGD) and PAP2b/VCIP derived peptides to inhibit morphogenic differentiation of endothelial cells.
• the vessels formed at the end of 24 hours of culture in 3D collagen were considered "pre-formed vessels", while capillaries formed after 24 hours of culture were considered as "new capillaries”.
• Anti- ⁇ v ⁇ 3 monoclonal antibodies only reduced the number of pre-formed capillaries by less than 50%, suggesting that other cell surface proteins, such as the fibronectin-binding integrin ⁇ 5 ⁇ i and collagen/laminin binding integrins might also play a role in capillary morphogenesis. Indeed, anti- ⁇ _ integrin subunit monoclonal antibodies inhibited preformed interconnections and reduced the number of capillaries by -60-70% ( Figure 22).
• anti- ⁇ 2 ⁇ integrin monoclonal antibodies reduced the number of capillaries by 45% to 60% over the period of 36 to 72 hours, suggesting that the collagen-binding ⁇ 2 ⁇ i integrin was the major receptor involved in signaling from the type I collagen matrix used in this assay (data not shown). It remains possible that type I collagen receptors ⁇ 10 ⁇ i and integrins could also play a role. No two monoclonal antibodies were added together, since it has been reported that a 5 ⁇ and ⁇ v ⁇ 3 integrin antibodies together induce complete collapse and regression of tubules in vitro.
• Anti-PAP2b-RGD (anti-VCIP-RGD) monoclonal antibodies inhibited the formation of new capillaries, reducing the number of capillaries by -45% to 55% after 60 to 72 hours.
• Anti-KDR and anti- VE-cadherin monoclonal antibodies reduced the number of capillaries by 45% to 75% over the period of 36 to 72 hours. It is likely that anti-KDR and anti- VE-cadherin monoclonal antibodies blocked different signaling pathways. Whereas VEGF-induced KDR signaling is required for cell proliferation, survival, and differentiation, as well as vascular permeability, VE-Cadherin is required for the maintenance of cell-cell junctions and for cell polarization. Antibodies that affect any one aspect of the endothelial cell activation and differentiation pathway are likely to inhibit capillary morphogenesis in vitro.
• Wary et al. The adaptor protein She couples a class of integrins to the control of cell cycle progression. Cell 87:733-743 (1996). Wary et al., A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell 94:625-634 (1998). Wary et al., Biochemical analysis of integrin-mediated She signaling. In Methods in Molecular Biology, Howlett ed., Humana Press, Totowa, NJ, 35-49 (1999a). Wary et al., Specificity of integrin signaling. In Signaling through Cell Adhesion Molecules,

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