Targeting Pancreatic Islets with Phage Display Assisted by Laser Pressure Catapult Microdissection
2005; Elsevier BV; Volume: 166; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)62283-3
ISSN1525-2191
AutoresVirginia Yao, Michael G. Ozawa, Martin Trepel, Wadih Arap, Donald M. McDonald, Renata Pasqualini,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoHeterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature. Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature. Peptides that target vascular receptors by in vivo phage display have been successfully identified in the mouse and in a human subject.1Arap W Kolonin MG Trepel M Lahdenranta J Cardó-Vila M Giordano RJ Mintz PJ Ardelt PU Yao VJ Vidal CI Chen L Flamm A Valtanen H Weavind LM Hicks ME Pollock RE Botz GH Bucana CD Koivunen E Cahill D Troncoso P Baggerly KA Pentz RD Do KA Logothetis CJ Pasqualini R Steps toward mapping the human vasculature by phage display.Nat Med. 2002; 8: 121-127Crossref PubMed Scopus (505) Google Scholar, 2Kolonin MG Pasqualini R Arap W Teratogenicity induced by targeting a placental immunoglobulin transporter.Proc Natl Acad Sci USA. 2002; 99: 13055-13060Crossref PubMed Scopus (35) Google Scholar, 3Essler M Ruoslahti E Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature.Proc Natl Acad Sci USA. 2002; 99: 2252-2257Crossref PubMed Scopus (170) Google Scholar, 4Rajotte D Ruoslahti E Membrane dipeptidase is the receptor for a lung-targeting peptide identified by in vivo phage display.J Biol Chem. 1999; 274: 11593-11598Crossref PubMed Scopus (152) Google Scholar, 5Pasqualini R Koivunen E Ruoslahti E Alpha v integrins as receptors for tumor targeting by circulating ligands.Nat Biotechnol. 1997; 15: 542-546Crossref PubMed Scopus (709) Google Scholar, 6Pasqualini R Ruoslahti E Organ targeting in vivo using phage display peptide libraries.Nature. 1996; 380: 364-366Crossref PubMed Scopus (1057) Google Scholar The resultant peptide sequences illustrate the heterogeneous nature of vascular receptors from organ to organ.1Arap W Kolonin MG Trepel M Lahdenranta J Cardó-Vila M Giordano RJ Mintz PJ Ardelt PU Yao VJ Vidal CI Chen L Flamm A Valtanen H Weavind LM Hicks ME Pollock RE Botz GH Bucana CD Koivunen E Cahill D Troncoso P Baggerly KA Pentz RD Do KA Logothetis CJ Pasqualini R Steps toward mapping the human vasculature by phage display.Nat Med. 2002; 8: 121-127Crossref PubMed Scopus (505) Google Scholar, 7Pasqualini R Arap W McDonald DM Probing the structural and molecular diversity of tumor vasculature.Trends Mol Med. 2002; 8: 563-571Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 8Trepel M Arap W Pasqualini R In vivo phage display and vascular heterogeneity: implications for targeted medicine.Curr Opin Chem Biol. 2002; 6: 399-404Crossref PubMed Scopus (181) Google Scholar In addition to this difference, many organs such as the adrenal gland, kidney, pancreas, and brain contain functionally distinct regions that are embedded within the tissue and exhibit a unique vascular organization that suggests differential molecular vascular addresses within the same organ. Extraction of such functionally specialized regions from excised organs using conventional biochemical methods may fail to recover ligand-receptor pairs. For example, physical separation of a specific site from the surrounding tissues after in vivo phage library biopanning may disrupt the complex between the receptor and the bound peptide-phage by mechanical manipulations and/or nonphysiological buffer conditions. Moreover, protease inhibitor cocktails may alleviate phage degradation by endogenous proteases in crude tissue homogenates, however the cost effectiveness of their use may be prohibitive in a complex subcellular fractionation scheme that may ultimately diminish final yields. We set out to determine whether phage that home to a functionally specialized region within an organ could be isolated using a combination of in vivo phage display with fluorescence laser microbeam microdissection and laser pressure catapulting, hereafter referred to as laser pressure catapult microdissection (LPCM).9Schutze K Lahr G Identification of expressed genes by laser-mediated manipulation of single cells.Nat Biotechnol. 1998; 16: 737-742Crossref PubMed Scopus (375) Google Scholar We investigated the vascular heterogeneity10Dib SA Vardi P Bonner-Weir S Eisenbarth GS Selective localization of factor VIII antigenicity to islet endothelial cells and expression of class II antigens by normal human pancreatic ductal epithelium.Diabetes. 1988; 37: 482-487Crossref PubMed Scopus (12) Google Scholar of the murine pancreatic islet for the following rationale: islets perform specialized functions that are biologically and clinically relevant, and the islet vasculature is readily identifiable from that of the surrounding acinar pancreas.11Beck JSP Berg BN The circulatory pattern in the islands of Langerhans.Am J Pathol. 1931; 7: 31-35PubMed Google Scholar Applications of this study include development of specific peptide-based targeting therapies that recognize functionally distinct intravascular networks that encompass a broad range of human diseases. Eight-week-old wild-type C57BL/6 male mice were purchased from Harlan (Indianapolis, IN). Male C57BL/6/RIP-Tag2 transgenic mice produce spontaneous pancreatic islet tumors12Hanahan D Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.Nature. 1985; 315: 115-122Crossref PubMed Scopus (1082) Google Scholar and were genotyped using tail-tip DNA by the polymerase chain reaction (PCR). Mice were housed under barrier conditions at the animal care facility at either The University of Texas, M. D. Anderson Cancer Center (MDACC) or the University of California, San Francisco (UCSF). All experimental procedures were approved by the Institutional Animal Care and Use Committees at MDACC and UCSF. 109 transforming units of a cyclic CX7C peptide phage library1Arap W Kolonin MG Trepel M Lahdenranta J Cardó-Vila M Giordano RJ Mintz PJ Ardelt PU Yao VJ Vidal CI Chen L Flamm A Valtanen H Weavind LM Hicks ME Pollock RE Botz GH Bucana CD Koivunen E Cahill D Troncoso P Baggerly KA Pentz RD Do KA Logothetis CJ Pasqualini R Steps toward mapping the human vasculature by phage display.Nat Med. 2002; 8: 121-127Crossref PubMed Scopus (505) Google Scholar was intravenously injected via the tail vein of Avertin (2,2,2-tribromoethanol, 0.015 to .017 mg/g injected intraperitoneally; Sigma-Aldrich Corp., St. Louis, MO) anesthetized C57BL/6 male mice,13Papaioannou VE Fox JG Efficacy of tribromoethanol anesthesia in mice.Lab Anim Sci. 1993; 43: 189-192PubMed Google Scholar and allowed to circulate for 6 minutes while maintaining the body temperature of the mice at 37°C with a heating pad. The pancreas was removed, weighed, and homogenized in ice-cold Dulbecco's modified Eagle's medium containing Earle salts (MDACC cell culture facility) supplemented with 1% bovine serum albumin, 1 mmol/L 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 10 μg/ml aprotinin, 1 μg/ml leupeptin (Calbiochem, San Diego, CA). The pancreas homogenate was resuspended, rinsed 3× in the same buffer and phage were recovered by infection of the bacterial host, K91.14Smith GP Scott JK Libraries of peptides and proteins displayed on filamentous phage.Methods Enzymol. 1993; 217: 228-257Crossref PubMed Scopus (709) Google Scholar Phage-infected K91 recovered in 10 ml of Luria-Bretani (LB)/kanamycin (Kan, 100 mg/L)/tetracycline (Tet, 0.2 mg/L) for 20 minutes in the dark at room temperature. Aliquots from each culture were plated onto LB/Kan/Tet (40 mg/L) agar plates and incubated overnight in the dark at 37°C. Two hundred and forty-six bacterial colonies were each grown overnight in 5 ml of LB/Kan (100 mg/L)/Tet (40 mg/L) at 37°C in the dark with agitation. Phage were purified from pooled overnight cultures and infective titers were determined using established protocols.15Pasqualini R Arap W Rajotte D Ruoslahti E In vivo selection of phage display libraries.in: Barbas III, CF DR Burton JK Scott GJ Silverman Phage Display A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor2001: 22-29Google Scholar Three hundred colonies were picked from the second round, of which 96 colonies were randomly selected for colony PCR and automated sequencing of the peptide insert at the MDACC Sequencing Core Facility. For the third panning round, intravenous injection of 109 transforming units of purified, amplified round II phage was followed by an injection of 50 μg of fluorescein isothiocyanate (FITC)-lectin (Vector Laboratories, Inc., Burlingame, CA). Mice were infused through the right ventricle with 3 ml of 37°C Dulbecco's modified Eagle's medium. The inferior vena cava was cut for the outlet. The pancreas was harvested, frozen at −80°C in Tissue Tek O.C.T. compound (Sakura, Torrance, CA), and 14-μm sections were cut and placed onto prepared microscope slides for LPCM (MDACC Veterinary Technical Services). The total amount of phage recovered from the round III pancreas was determined by extrapolating the number of bacterial colonies recovered from four, 14-μm thawed round III pancreatic sections to the final thickness (5 mm) of the frozen tissue block. One hundred and twenty islet sections were microdissected and catapulted from 14-μm frozen pancreas sections using the positioning and ablation with laser microbeams system (P.A.L.M. Mikrolaser Technologie GmbH, Bernried, Germany) fitted to a Zeiss Axiovert 135 microscope with Fluar objectives and a fluorescence module using the manufacturer's suggested laser-cutting and focus energy. Excised islet sections were catapulted into 30 μl of either 1 mmol/L ethylenediaminetetraacetic acid, pH 8.0 (Sigma-Aldrich Corp.) for PCR amplification, or a protease inhibitor cocktail consisting of 1 mmol/L AEBSF 20 μg/ml aprotinin, 10 μg/L leupeptin, 1 mmol/L elastase inhibitor I, 0.1 mmol/L Na-Tosyl-Phe chloromethyl ketone, 1 nmol/L pepstatin A in phosphate buffered saline (PBS), pH 7.4, for phage recovery by bacterial infection (see below). All protease inhibitors were purchased from Calbiochem. Catapulted islet sections were incubated with 1 ml of stationary phase K91 for 2 hours at room temperature with gentle agitation. Each culture was transferred to 1.2 ml of LB/Kan (100 mg/L)/Tet (0.2 mg/L) and incubated in the dark at room temperature for 40 minutes. The tetracycline concentration was increased to 40 mg/L, and the cultures were incubated overnight at 37°C with agitation. Cultures were plated out onto LB/Kan/Tet agar plates to obtain single colonies. Peptide insert sequences were amplified by colony PCR and sequenced at the MDACC Sequencing Core Facility. Peptide insert sequences were initially amplified by PCR from catapulted islet sections using the fUSE5 phage forward primer,16Koivunen E Arap W Rajotte D Lahdenranta J Pasqualini R Identification of receptor ligands with phage display peptide libraries.J Nucl Med. 1999; 40: 883-888PubMed Google Scholar and reverse primer, 5′-CCCTCATAGTTAGCGTAACGATCT-3′. Flanking SfiI restriction sites were added to each peptide insert sequence by a second PCR amplification by using the forward Sfi library primer 5′-CACTCGGCCGACGGGC-3′, and reverse SfiI primer: 5′-CAGTTTCGGCCCCAGCGGCCC-3′ for ligation into SfiI-digested CsCl2-purified fUSE5 genomic DNA.17Bonnycastle LLC Menendez A Scott JK General phage methods, phage preparation on CsCl gradients.in: Barbas III, CF DR Burton JK Scott GJ Silverman Phage Display A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor2001: 15.11-15.13Google Scholar All primers were purchased from Genosys (The Woodlands, TX). Ligation products were electroporated into MC1061 bacterial hosts,14Smith GP Scott JK Libraries of peptides and proteins displayed on filamentous phage.Methods Enzymol. 1993; 217: 228-257Crossref PubMed Scopus (709) Google Scholar and plated onto LB/streptomycin (100 mg/L)/Tet (40 mg/L) agar plates. Single colonies were subjected to colony PCR and sequenced at the MDACC Sequencing Core Facility. Single-stranded CVSNPRWKC, CHVLWSTRC, and fd-tet phage DNA were electroporated into electrocompetent MC1061 bacterial hosts, amplified phage were purified from overnight cultures and titered using established protocols.15Pasqualini R Arap W Rajotte D Ruoslahti E In vivo selection of phage display libraries.in: Barbas III, CF DR Burton JK Scott GJ Silverman Phage Display A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor2001: 22-29Google Scholar Peptide insert sequences were verified by automated sequencing (ELIM Biopharmaceuticals, Hayward, CA). Phage preparations were used within 24 hours of preparation. Before injection, 109 transforming units of CVSNPRWKC, CHVLWSTRC, and fd-tet phage preparations were diluted into sterile PBS to a final volume of 200 μl. Fifty μg of polyclonal goat anti-mouse EphA4 antibodies (R&D Systems, Minneapolis, MN) and normal goat IgG (Jackson ImmunoResearch, West Grove, PA) were diluted with sterile PBS and filtered through a 0.22-μm filter. Phage, EphA4 antibodies, or goat IgGs were injected intravenously into male C57BL/6 or RIP-Tag2 mice, allowed to circulate for 6 minutes, and then the mice were systemically perfused with 1% paraformaldehyde in PBS, pH 7.4 (PFA/PBS) through the left ventricle for 3 minutes at 120 mmHg. The inferior vena cava was cut as an outlet for the fixative. Organs were removed, incubated in 1% PFA/PBS for 1 to 2 hours, embedded overnight at 4°C in 30% sucrose/PBS/0.01% Thimerosol (Sigma-Aldrich Corp.), and frozen in O.C.T. on dry ice. All steps were performed at room temperature unless otherwise indicated. Eighty-μm frozen tissue sections were air dried, rinsed 2× for 5 minutes each with PBS, 1× with PBS/1% Triton X-100, and then blocked with 5% normal mouse serum (Jackson ImmunoResearch) in PBS/1% Triton X-100 for 1 hour. For phage immunostaining, sections were incubated in a 0.22-μm filtered primary antibody solution containing monoclonal rat anti-mouse CD31 antibody (1:500; Pharmingen, San Diego, CA), polyclonal rabbit fd-bacteriophage antibody (1:5000; Sigma-Aldrich Corp.), and 5% normal mouse serum in PBS/1% Triton X-100 for at least 14 hours. Sections were rinsed 3× for 5 minutes each with PBS/1% Triton X-100. Rinsed sections were incubated in a 0.22-μm filtered solution containing mouse FITC-conjugated anti-rat IgG (1:200, Jackson ImmunoResearch), mouse Cy3-conjugated anti-rabbit IgG (1:400, Jackson ImmunoResearch), and 5% normal mouse serum in PBS/1% Triton X-100 for 2 hours. Prepared sections containing EphA4 antibody were incubated with monoclonal rat anti-mouse CD31 in 5% normal mouse serum in PBS/1% Triton X-100, rinsed, and incubated with mouse FITC-conjugated anti-rat IgG, mouse Cy3-conjugated anti-goat IgG (1:400, Jackson ImmunoResearch), and 5% normal mouse serum in PBS/1% Triton X-100. After immunostaining, all sections were rinsed 3×, 10 minutes each, with PBS/1% Triton X-100, fixed for 5 minutes with 4% PFA/PBS, rinsed 3× with PBS for 10 minutes each, and mounted with Vectashield (Vector Laboratories, Inc.). Fluorescence images were acquired using an externally coded, three-chip charge-couple device camera (CoolCam; SciMeasure Analytical Systems, Atlanta, GA) fitted on a Zeiss Axiophot fluorescence microscope with Fluar objectives or with a Zeiss LSM 510 laser scanning confocal microscope with krypton-argon and helium-neon lasers (Carl Zeiss, Jena, Germany) and analyzed with the LSM 510 software (version 3.2.2). To prepare tissues for LPCM, we performed three rounds of in vivo phage display starting with a cyclic CX7C phage library (Figure 1A).15Pasqualini R Arap W Rajotte D Ruoslahti E In vivo selection of phage display libraries.in: Barbas III, CF DR Burton JK Scott GJ Silverman Phage Display A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor2001: 22-29Google Scholar Colony counts of phage recovered from excised pancreata showed increased selectivity of targeting peptides homing to the pancreas with each successive panning round (Figure 1B). Islets were readily distinguished from the surrounding acinar pancreas by their characteristic vascular organization as revealed by fluorescein isothiocyanate-conjugated Lycopersicon esculentum lectin (FITC-lectin)-labeled blood vessels (Figure 1C).18Thurston G McLean JW Rizen M Baluk P Haskell A Murphy TJ Hanahan D McDonald DM Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice.J Clin Invest. 1998; 101: 1401-1413Crossref PubMed Google Scholar Phage recovery from LPCM islet sections was achieved by two routes, bacterial infection15Pasqualini R Arap W Rajotte D Ruoslahti E In vivo selection of phage display libraries.in: Barbas III, CF DR Burton JK Scott GJ Silverman Phage Display A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor2001: 22-29Google Scholar and subcloning nested PCR-amplified peptide insert sequences into the fUSE5 phage genome (Figure 1C). Bacterial infection of phage recovered from catapulted islet sections yielded eight bacterial clones from which seven unique peptide sequences were obtained (Table 1). Conversely, subcloning PCR-amplified peptide insert sequences from catapulted islets into the fUSE5 phage genome yielded hundreds of transformed bacterial clones, however, sequence analyses of 80 randomly selected clones yielded 11 unique sequences. Identified peptide sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information web site (http://www.ncbi.nlm.nih.gov/BLAST) to search for putative homologous proteins (Table 1). Peptides CVPRRWDVC and CQHTSGRGC exhibited partial sequence identity to the laminin β-2 chain at positions 197 to 201 and 1088 to 1092, respectively.19Durkin ME Gautam M Loechel F Sanes JR Merlie JP Albrechtsen R Wewer UM Structural organization of the human and mouse laminin beta2 chain genes, and alternative splicing at the 5′ end of the human transcript.J Biol Chem. 1996; 271: 13407-13416Crossref PubMed Scopus (27) Google Scholar Peptides CVSNPRWKC and CHVLWSTRC exhibited partial sequence identity to ephrin-A2 and ephrin-A4 which are glycosylphosphatidylinositol-anchored proteins that bind to EphA-type receptors.20Gale NW Yancopoulos GD Ephrins and their receptors: a repulsive topic?.Cell Tissue Res. 1997; 290: 227-241Crossref PubMed Scopus (136) Google ScholarTable 1Peptide Sequences Expressed by Phage Recovered from Pancreatic Islet SectionsIslet-homing peptide sequence*Amino acid residues in bold share sequence identity, including conservative replacements, to the listed murine proteins.Colonies recovered by K91 infectionColonies recovered by PCR/subcloningMurine protein sharing complete/partial sequence identitySWISSPROT accession numberCVSNPRWKC10Ephrin-A2, Ephrin-A4P52801, O08542CQHTSGRGC10S-laminin β2 chainQ61292CRARGWLLC10α3 integrinQ62470CGGVHALRC10PDGF receptor βP05622CFNRTWIGC10Frizzled-1O70421CSRGPAWGC10Bone morphogenetic protein 3bP97737CVPRRWDVC20S-laminin β2 chainQ61292CWSRGQGGC021Endothelin-1 receptorQ61614CHVLWSTRC011Ephrin-A2, Eprhrin-A4P52801, O08542CLGLLMAGC011VE-cadherinP55284CMSSPGVAC09Insulin receptor substrate 1P35569CLASGMDAC08Tumor necrosis factor-αP06804CHDERTGRC06Angio-associated migratory proteinQ8K2C1CAHHALMEC05Adiponectin receptor 1, 2Q91VH1, Q8BQS5CMQGAATSC04Grb10Q60760CVRDLLTGC03α4 integrinQ00651CMQGARTSC01α2C-adrenergic receptorQ01337CGGLVAVGC01β7 integrinP26011* Amino acid residues in bold share sequence identity, including conservative replacements, to the listed murine proteins. Open table in a new tab Precedence for vascular expression of both classes of Eph receptors and ephrin ligands has been documented. Mouse knockout studies have shown interactions between the EphB2, B3, and B4 receptors, and their corresponding ligands, ephrinB1 and B2, are necessary for vasculogenesis and angiogenesis.21Gale NW Baluk P Pan L Kwan M Holash J DeChiara TM McDonald DM Yancopoulos GD Ephrin-B2 selectively marks arterial vessels and neovascularization sites in the adult, with expression in both endothelial and smooth-muscle cells.Dev Biol. 2001; 230: 151-160Crossref PubMed Scopus (325) Google Scholar Ephrin-A1 expression is induced by tumor necrosis factor-α in human umbilical vein endothelial cells and its binding to the EphA2 receptor results in receptor autophosphorylation.22Pandey A Shao H Marks RM Polverini PJ Dixit VM Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF-alpha-induced angiogenesis.Science. 1995; 268: 567-569Crossref PubMed Scopus (346) Google Scholar Ephrin-A1 induces angiogenesis in rat corneal pocket assays via endothelial cell migration rather than by endothelial cell proliferation.22Pandey A Shao H Marks RM Polverini PJ Dixit VM Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF-alpha-induced angiogenesis.Science. 1995; 268: 567-569Crossref PubMed Scopus (346) Google Scholar The EphA2 receptor and cognate ephrin-A1 ligand are expressed in RIP-Tag2 murine pancreatic islet tumor endothelium suggesting a role for these proteins in cancer.23Brantley DM Cheng N Thompson EJ Lin Q Brekken RA Thorpe PE Muraoka RS Cerretti DP Pozzi A Jackson D Lin C Chen J Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo.Oncogene. 2002; 21: 7011-7026Crossref PubMed Scopus (291) Google Scholar In vitro phage display identified two peptides, SWLAYPGAVSYR and YSAYPDSVPMMS, that mimic ephrin A-type ligands because both peptides bind to immobilized EphA2-Fc protein and to the EphA2 receptor expressed on human umbilical vein endothelial cells and MDA-MB-435 cultured human breast carcinoma cells.24Koolpe M Dail M Pasquale EB An ephrin mimetic peptide that selectively targets the EphA2 receptor.J Biol Chem. 2002; 277: 46974-46979Crossref PubMed Scopus (181) Google Scholar We mapped the location of the CVSNPRWKC and CHVLWSTRC peptides to the aligned primary structures of murine ephrin-A2 and ephrin-A4, and human ephrin-A2 using DIALIGN 2.2.125Morgenstern B DIALIGN 2: improvement of the segment-to-segment approach to multiple sequence alignment.Bioinformatics. 1999; 15: 211-218Crossref PubMed Scopus (574) Google Scholar (Figure 2). Murine and human ephrin-A2 share 89% sequence identity, whereas the murine ephrins A2 and A4 share 43% sequence identity. Secondary structure predictions of the glycosylphosphatidylinositol-linked A-type ephrins were extrapolated from the crystal structures of the ephrin-B2 extracellular domain26Toth J Cutforth T Gelinas AD Bethoney KA Bard J Harrison CJ Crystal structure of an ephrin ectodomain.Dev Cell. 2001; 1: 83-92Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar and the EphB2-ephrin-B2 complex.27Himanen JP Rajashankar KR Lackmann M Cowan CA Henkemeyer M Nikolov DB Crystal structure of an Eph receptor-ephrin complex.Nature. 2001; 414: 933-938Crossref PubMed Scopus (280) Google Scholar Peptide CVSNPRWKC mapped to the murine ephrin A2 N-terminus at amino acid residues 41 to 44 with a conservative replacement of a Trp residue in the peptide sequence for Phe45 in ephrin-A2. Structurally, the SNPRW peptide sequence lies between the A′ and B anti-parallel β-strands and appears to be well conserved among the A-type ephrins from mice, rats, and humans.26Toth J Cutforth T Gelinas AD Bethoney KA Bard J Harrison CJ Crystal structure of an ephrin ectodomain.Dev Cell. 2001; 1: 83-92Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 27Himanen JP Rajashankar KR Lackmann M Cowan CA Henkemeyer M Nikolov DB Crystal structure of an Eph receptor-ephrin complex.Nature. 2001; 414: 933-938Crossref PubMed Scopus (280) Google Scholar In addition, this sequence is C-terminal from a putative glycosylation site at Asn38 that is analogous to Asn39 in ephrins B2 and B3, and is conserved in ephrins A1, A2, A4, and A5.26Toth J Cutforth T Gelinas AD Bethoney KA Bard J Harrison CJ Crystal structure of an ephrin ectodomain.Dev Cell. 2001; 1: 83-92Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 27Himanen JP Rajashankar KR Lackmann M Cowan CA Henkemeyer M Nikolov DB Crystal structure of an Eph receptor-ephrin complex.Nature. 2001; 414: 933-938Crossref PubMed Scopus (280) Google Scholar Asn38 has been proposed to be involved in a weaker affinity ligand-receptor tetramerization interface.28Himanen JP Nikolov DB Eph signaling: a structural view.Trends Neurosci. 2003; 26: 46-51Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar Peptide CHVLWSTRC mapped to the unstructured C-terminus of murine ephrinA2 at residues 202 to 205. The VLWS peptide sequence also mapped to the signal sequence of murine ephrin-A4 at positions 10 to 12, and showed a weaker sequence identity in the D β-strand at positions 78 to 81. We verified CVSNPRWKC and CHVLWSTRC phage binding to the C57BL/6 pancreatic islet vasculature by immunohistochemistry (Figure 3). Intravenously injected CVSNPRWKC phage (Figure 3A), and CHVLWSTRC phage (Figure 3B) localized predominantly to normal pancreatic islet blood vessels, whereas the insertless control phage, fd-tet, did not bind to either the acinar or islet vasculature (Figure 3C). Because unregulated overexpression of proteins is a common feature in tumors,29Bennasroune A Gardin A Aunis D Cremel G Hubert P Tyrosine kinase receptors as attractive targets of cancer therapy.Crit Rev Oncol Hematol. 2004; 50: 23-38Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar we wanted to determine whether localization of the islet homing peptide-phage, CVSNPRWKC and CHVLWSTRC, was increased in angiogenic tumor blood vessels. We compared the binding of CVSNPRWKC and CHVLWSTRC phage to the negative control phage, fd-tet, in pancreatic islet tumors from RIP-Tag2 transgenic mice12Hanahan D Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.Nature. 1985; 315: 115-122Crossref PubMed Scopus (1082) Google Scholar (Figure 4). Insertless fd-tet phage neither accumulated in islet tumor blood vessels nor in blood vessels of the surrounding normal acinar pancreas (Figure 4, A and B). Conversely, CHVLWSTRC phage were abundant in tumor blood vessels that were delineated by phage immunoreactivity (Figure 4, C and D; encircled large tumor). Binding of CHVLWSTRC phage was reduced in blood vessels of a smaller premalignant angiogenic islet (Figure 4C, encircled right islet; and Figure 4D, arrow), whereas phage binding was absent in acinar blood vessels (Figure 4, C and D; asterisk). Similarly, CVSNPRWKC phage immunoreactivity was robust in blood vessels of large islet tumor with insignificant phage immunoreactivity in acinar blood vessels (Figure 4E). CVSNPRWKC or CHVLWSTRC phage did not bind to blood vessels of the brain, lung, or mesenteric lymph nodes, whereas the liver and spleen contained both peptide-expressing phage and fd-tet phage as expected (data not shown).6Pasqualini R Ruoslahti E Organ targeting in vivo using phage display peptide libraries.Nature. 1996; 380: 364-366Crossref PubMed Scopus (1057) Google Scholar, 15Pasqualini R Arap W Rajotte D Ruoslahti E In vivo selection of phage display libraries.in: Barbas III, CF DR Burton JK Scott GJ Silverman Phage Display A Laboratory Manual. Cold Spring Harbor Laboratory Press, Co
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