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• W2004865667 abstract "Neprilysin is a cell surface peptidase that catalytically inactivates neuropeptide substrates and functions as a tumor suppressor via its enzymatic function and multiple protein-protein interactions. We investigated whether neutral endopeptidase could inhibit angiogenesis in vivo utilizing a murine corneal pocket angiogenesis model and found that it reduced fibroblast growth factor-2-induced angiogenesis by 85% (p < 0.0151515156) but had no effect on that of vascular endothelial growth factor. Treatment with recombinant neprilysin, but not enzymatically inactive neprilysin, resulted in a slight increase in basic fibroblast growth factor electrophoretic mobility from proteolytic cleavage between amino acids Leu-135 and Gly-136, which was inhibited by the neutral endopeptidase inhibitor CGS24592 and heparin. Cleavage kinetics were rapid, comparable with that of other known neprilysin substrates. Functional studies involving neprilysin-expressing vascular endothelial cells demonstrated that neutral endopeptidase inhibition significantly enhanced fibroblast growth factor-mediated endothelial cell growth, capillary array formation, and signaling, whereas exogenous recombinant neprilysin inhibited signaling. Recombinant constructs confirmed that cleavage products neither promoted capillary array formation nor induced signaling. Moreover, mutation of the cleavage site resulted in concomitant loss of cleavage and increased the potency of fibroblast growth factor-2 to induce capillary array formation. These data indicate that neprilysin proteolytically inactivates fibroblast growth factor-2, resulting in negative regulation of angiogenesis. Neprilysin is a cell surface peptidase that catalytically inactivates neuropeptide substrates and functions as a tumor suppressor via its enzymatic function and multiple protein-protein interactions. We investigated whether neutral endopeptidase could inhibit angiogenesis in vivo utilizing a murine corneal pocket angiogenesis model and found that it reduced fibroblast growth factor-2-induced angiogenesis by 85% (p < 0.0151515156) but had no effect on that of vascular endothelial growth factor. Treatment with recombinant neprilysin, but not enzymatically inactive neprilysin, resulted in a slight increase in basic fibroblast growth factor electrophoretic mobility from proteolytic cleavage between amino acids Leu-135 and Gly-136, which was inhibited by the neutral endopeptidase inhibitor CGS24592 and heparin. Cleavage kinetics were rapid, comparable with that of other known neprilysin substrates. Functional studies involving neprilysin-expressing vascular endothelial cells demonstrated that neutral endopeptidase inhibition significantly enhanced fibroblast growth factor-mediated endothelial cell growth, capillary array formation, and signaling, whereas exogenous recombinant neprilysin inhibited signaling. Recombinant constructs confirmed that cleavage products neither promoted capillary array formation nor induced signaling. Moreover, mutation of the cleavage site resulted in concomitant loss of cleavage and increased the potency of fibroblast growth factor-2 to induce capillary array formation. These data indicate that neprilysin proteolytically inactivates fibroblast growth factor-2, resulting in negative regulation of angiogenesis. Neprilysin (neutral endopeptidase 24.11, CD10) is a 90-110-kDa cell surface peptidase normally expressed by a variety of tissues, including epithelial cells of the prostate, kidney, intestine, endometrium, adrenal glands, and lung. This enzyme cleaves peptide bonds on the amino side of hydrophobic amino acids and inactivates a variety of physiologically active peptides, including atrial natriuretic factor, substance P, bradykinin, oxytocin, Leu- and Met-enkephalins, neurotensin, bombesin, endothelin-1, and β-amyloid. Loss or a decrease in neprilysin expression has been reported in a variety of malignancies, including renal cancer, invasive bladder cancer, poorly differentiated stomach cancer, small cell and nonsmall cell lung cancers, endometrial cancer, and prostate cancer (1Papandreou C.N. Usmani B. Geng Y. Bogenrieder T. Freeman R. Wilk S. Finstad C.L. Reuter V.E. Powell C.T. Scheinberg D. Magill C. Scher H.I. Albino A.P. Nanus D.M. Nat. Med. 1998; 4: 50-57Crossref PubMed Scopus (243) Google Scholar, 2Osman I. Yee H. Taneja S.S. Levinson B. Zeleniuch-Jacquotte A. Chang C. Nobert C. Nanus D.M. Clin. Cancer Res. 2004; 10: 4096-4100Crossref PubMed Scopus (47) Google Scholar). Reduced expression of cell surface peptidases such as neprilysin results in the accumulation of higher peptide concentrations that mediate neoplastic progression (3Nanus D.M. Clin. Cancer Res. 2003; 9: 6307-6309PubMed Google Scholar).Using prostate cancer as a model to study the involvement of neprilysin in malignancy, we have demonstrated the following. 1) Neprilysin protein expression is absent in nearly 50% of primary prostate cancers (2Osman I. Yee H. Taneja S.S. Levinson B. Zeleniuch-Jacquotte A. Chang C. Nobert C. Nanus D.M. Clin. Cancer Res. 2004; 10: 4096-4100Crossref PubMed Scopus (47) Google Scholar). 2) Neprilysin inhibits neuropeptide-mediated cell growth, cell migration, and ligand-independent activation of the insulin-like growth factor-1 receptor leading to Akt phosphorylation (1Papandreou C.N. Usmani B. Geng Y. Bogenrieder T. Freeman R. Wilk S. Finstad C.L. Reuter V.E. Powell C.T. Scheinberg D. Magill C. Scher H.I. Albino A.P. Nanus D.M. Nat. Med. 1998; 4: 50-57Crossref PubMed Scopus (243) Google Scholar, 4Sumitomo M. Milowsky M.I. Shen R. Navarro D. Dai J. Asano T. Hayakawa M. Nanus D.M. Cancer Res. 2001; 61: 3294-3298PubMed Google Scholar). 3) Neprilysin can inhibit cell migration independently of its catalytic activity via protein-protein interaction of its cytoplasmic domain with tyrosine-phosphorylated Lyn kinase, which then binds the p85 subunit of phosphatidylinositol 3-kinase resulting in an neprilysin-Lyn-phosphatidylinositol 3-kinase protein complex. This complex competitively blocks the interaction between focal adhesion kinase and phosphatidylinositol 3-kinase (5Sumitomo M. Shen R. Walburg M. Dai J. Geng Y. Navarro D. Boileau G. Papandreou C.N. Giancotti F.G. Knudsen B. Nanus D.M. J. Clin. Investig. 2000; 106: 1399-1407Crossref PubMed Scopus (124) Google Scholar). 4) Neprilysin directly binds to ezrin/radixin/moesin proteins resulting in decreased binding of ezrin/radixin/moesin proteins to the hyaluronan receptor CD44, such that cells expressing neprilysin demonstrate decreased cell adhesion and cell migration (6Iwase A. Shen R. Navarro D. Nanus D.M. J. Biol. Chem. 2004; 279: 11898-11905Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). 5) Neprilysin directly interacts with the PTEN tumor suppressor protein, recruiting endogenous PTEN to the cell membrane, leading to prolonged PTEN protein stability and increased PTEN phosphatase activity and resulting in a constitutive down-regulation of Akt activity (7Sumitomo M. Iwase A. Zheng R. Navarro D. Kaminetzky D. Shen R. Georgescu M.M. Nanus D.M. Cancer Cell. 2004; 5: 67-78Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). 6) Neprilysin expression inhibits tumorigenicity in an animal model of prostate cancer (8Dai J. Shen R. Sumitomo M. Goldberg J.S. Geng Y. Navarro D. Xu S. Koutcher J.A. Garzotto M. Powell C.T. Nanus D.M. Clin. Cancer Res. 2001; 7: 1370-1377PubMed Google Scholar). Taken together, these studies have demonstrated that neprilysin protein functions to suppress and inhibit many processes that contribute to neoplastic progression.Enzymatically active neprilysin is also expressed by vascular endothelial cells of venous and arterial origin (9Llorens-Cortes C. Huang H. Vicart P. Gasc J.M. Paulin D. Corvol P. J. Biol. Chem. 1992; 267: 14012-14018Abstract Full Text PDF PubMed Google Scholar). The neprilysin substrate endothelin-1 has previously been shown to act directly on endothelial cells via the ETB receptor to modulate different stages of neovascularization, including proliferation, migration, invasion, protease production, and morphogenesis, resulting in neovascularization in vivo (10Bagnato A. Spinella F. Trends Endocrinol. Metab. 2003; 14: 44-50Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Based on these observations, we investigated whether neprilysin also functions as an antagonist of angiogenesis. We report here that neprilysin is indeed anti-angiogenic in vivo, significantly inhibiting angiogenesis. Surprisingly, we demonstrate that neprilysin catalytically inactivates the potent angiogenic factor, fibroblast growth factor-2 (FGF-2). 3The abbreviations used are: FGF-2, fibroblast growth factor-2; ERK, extracellular-regulated kinase; FCS, fetal calf serum; GST, glutathione S-transferase; HSPG, heparan sulfate proteoglycan; HUVEC, human umbilical vein endothelial cell; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MTT, methyl-thiazol-tetrazolium; rNEP, recombinant neprilysin; tHBMEC, SV40-transduced human bone marrow microvascular endothelial cells; VEGF, vascular endothelial growth factor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 3The abbreviations used are: FGF-2, fibroblast growth factor-2; ERK, extracellular-regulated kinase; FCS, fetal calf serum; GST, glutathione S-transferase; HSPG, heparan sulfate proteoglycan; HUVEC, human umbilical vein endothelial cell; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MTT, methyl-thiazol-tetrazolium; rNEP, recombinant neprilysin; tHBMEC, SV40-transduced human bone marrow microvascular endothelial cells; VEGF, vascular endothelial growth factor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. This is the first report of an enzyme that specifically cleaves and inactivates FGF-2, resulting in inhibition of angiogenesis in vivo, further demonstrating the potent tumor-suppressive function of neprilysin.EXPERIMENTAL PROCEDURESCell Lines and Reagents—LNCaP cells were maintained in RPMI 1640 medium with 10% fetal calf serum (FCS) supplemented with penicillin (100 IU/ml) and streptomycin (100 μg/ml). Human umbilical vein endothelial cells (HUVEC) were isolated as previously described (11Jaffe E.A. Nachman R.L. Becker C.G. Minick C.R. J. Clin. Investig. 1973; 52: 2745-2756Crossref PubMed Scopus (5984) Google Scholar) and maintained in M199 medium (Invitrogen) supplemented with 10 units/ml heparin sodium, 10% FCS (Gemini), 2 mm l-glutamine, 100 μg/μl ECGS (Biomedical Technology) supplemented with penicillin and streptomycin. SV40-transduced human bone marrow microvascular endothelial cells (tHBMEC, kindly provided by Dr. Babette Weksler, Weill Medical College) were maintained in Dulbecco's modified Eagle's medium supplemented with 5% FCS as previously described (12Schweitzer K.M. Vicart P. Delouis C. Paulin D. Drager A.M. Langenhuijsen M.M. Weksler B.B. Lab. Investig. 1997; 76: 25-36PubMed Google Scholar). Vascular endothelial growth factor (VEGF) and growth factor-reduced Matrigel were purchased from BD Biosciences, and recombinant fibroblast growth factor-2 was purchased from Research Diagnostics, Inc.Proteolysis and Mass Spectrometry—Commercially available recombinant FGF-2 was incubated at a concentration of 5-13 μm with recombinant neprilysin (rNEP; Arris Pharmaceutical, Inc.) at a concentration of 0.4-1.0 μm in 100 mm Tris-HCl, pH 7.6, buffer for 1 h at 25°C in the presence or absence of 10 μm the specific neprilysin inhibitor CGS24592 (Novartis Pharmaceutical, Inc.) or 12 units/ml heparin sulfate. Reaction aliquots were analyzed by 14% SDS-PAGE with Coomassie Blue staining or by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; Micromass) in the mass spectroscopy core facility of Weill Medical College.GST-FGF-2 and Maltose-binding Protein-FGF-2 Fusion Proteins—Full-length human FGF-2 cDNA (kindly provided by Dr. Daniel Rifkin, New York University Medical Center) was used as template to amplify the entire FGF-2 cDNA, which was then subcloned into pGEX-2T (Amersham Biosciences) and pMAL-2Cx (New England Biolabs) vectors using PCR primers containing restriction sites to enable directional cloning. The following amplimers were used: 5′-ACCATGGCAGCCGGGAGCATC-3′ (sense) and 5′-ATATGAATTCTCAGCTCTTAGCAGACATGGAAGAAAG-3′ (antisense) for glutathioneS-transferase (GST) fusion proteins and 5′-ATGGCAGCCGGGAGCATC-3′ (sense) and 5′-CCCCAAGCTTTTAGCTCTTAGCAGACAT-3′ (antisense) for maltose-binding protein fusion proteins, as previously described (13Olsen E. Mohapatra S.S. Int. Arch. Allergy Immunol. 1992; 98: 343-348Crossref PubMed Scopus (11) Google Scholar). For GST constructs, the PCR product was then purified and digested with EcoRI and BamHI, generating the BamHI-EcoRI fragment corresponding to amino acids 136-155 and a BamHI-BamHI fragment corresponding to amino acids 1-135 of the FGF-2 protein. The restriction fragments were subcloned into pGEX-2T to generate GST fusion proteins with FGF-2 amino acids 1-155 (full-length), 1-135 (N-terminal neprilysin cleavage product), and 136-155 (C-terminal neprilysin cleavage product), and DNA sequencing was performed to confirm their accuracy. For maltose-binding protein constructs, the PCR product was digested with HindIII and XmnI and subcloned into the pMAL-2Cx vector. Fusion proteins of GST and maltose-binding protein with FGF-2 were expressed and purified from Escherichia coli BL21 cells using glutathione-agarose beads (Sigma) or amylase beads (New England Biolabs) as described (14Goodman Jr., O.B. Krupnick J.G. Gurevich V.V. Benovic J.L. Keen J.H. J. Biol. Chem. 1997; 272: 15017-15022Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Protein content of the beads and 10 mm glutathione eluants were determined by densitometric analysis of Coomassie-stained SDS-PAGE gels against bovine serum albumin standards (NIH ImageJ software). In some experiments FGF-2 was cleaved from amylose beads (1 mg of total fusion protein) by digestion with 2 units of Factor Xa in Tris-buffered saline buffer and affinity purified using heparin-Sepharose chromatography (15Casscells W. Speir E. Sasse J. Klagsbrun M. Allen P. Lee M. Calvo B. Chiba M. Haggroth L. Folkman J. Epstein S.E. J. Clin. Investig. 1990; 85: 433-441Crossref PubMed Scopus (143) Google Scholar). Site-directed mutants (L135A, G136A, and L135A/G136A) of the neprilysin cleavage site on FGF-2 were generated using a QuikChange mutagenesis kit (Stratagene) according to the manufacturer's instructions (mutagenic primers available upon request).Matrigel Capillary Array Formation Assay—Transduced human bone marrow microvascular endothelial cells (12Schweitzer K.M. Vicart P. Delouis C. Paulin D. Drager A.M. Langenhuijsen M.M. Weksler B.B. Lab. Investig. 1997; 76: 25-36PubMed Google Scholar) or HUVEC were plated in 96-well plates at a density of 15,000 cells/well in Dulbecco's modified Eagle's medium containing 5% FCS, penicillin, streptomycin, and l-glutamine over 50 μlof polymerized growth factor-reduced Matrigel (10 mg/ml). CGS24592 at a concentration of 0.5-10 nm, phosphoramidon (30 μm; Sigma), or an equivalent volume of dimethyl sulfoxide vehicle (Me2SO) was added to inhibit neprilysin activity for 2 h, followed by FGF-2 proteins at 0.3-50 nm concentrations. Cells were photographed after 4-18 h and measurements of capillary cord length obtained for multiple fields using ImageJ software and expressed as mean ± S.E. Statistical analyses of replicates were performed using unpaired two-tailed t testing (Prism Graph, GraphPad software).MTT Growth Assay—Transduced human bone marrow microvascular endothelial cells plated in 96-well plates at a density of 1000 cells/well in Dulbecco's modified Eagle's medium containing 5% FCS, penicillin, streptomycin, l-glutamine, and 600 pg/ml FGF-2 were treated with increasing concentrations of CGS24592. Growth assays with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were performed after 48-96 h as described (16Hoffman A.D. Engelstein D. Bogenrieder T. Papandreou C.N. Steckelman E. Dave A. Motzer R.J. Dmitrovsky E. Albino A.P. Nanus D.M. Clin. Cancer Res. 1996; 2: 1077-1082PubMed Google Scholar).Corneal Pocket Assay—Hydron (Hydro Med Sciences, Cranbury, NJ) and sucralfate (Teva Pharmaceuticals, North Wales, PA) pellets of <1 μl were formulated with combinations of FGF-2 (10 ng or 50 ng/pellet), VEGF (200 ng/pellet), and rNEP (50 or 100 ng/pellet) and implanted into corneas of C57BL/6 mice 0.5-1.0 mm from the limbus as described (17Volpert O.V. Lawler J. Bouck N.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6343-6348Crossref PubMed Scopus (157) Google Scholar). Angiogenesis was assessed by slit-lamp microscopy 5 days after implantation (18Simantov R. Febbraio M. Crombie R. Asch A.S. Nachman R.L. Silverstein R.L. J. Clin. Investig. 2001; 107: 45-52Crossref PubMed Scopus (89) Google Scholar). Statistical analysis of replicates was performed using a two-tailed unpaired t test (Prism Graph, GraphPad software). Studies were approved by the Institutional Animal Care Use Committee of Weill Medical College of Cornell University.Neprilysin Enzyme Assays—Neprilysin enzyme activity determinations were performed as previously described (1Papandreou C.N. Usmani B. Geng Y. Bogenrieder T. Freeman R. Wilk S. Finstad C.L. Reuter V.E. Powell C.T. Scheinberg D. Magill C. Scher H.I. Albino A.P. Nanus D.M. Nat. Med. 1998; 4: 50-57Crossref PubMed Scopus (243) Google Scholar) Briefly, subconfluent cells were rinsed in cold lysis buffer (50 mm Tris, pH 7.0, 150 mm NaCl) and lysed in lysis buffer containing 0.5% CHAPS (3-[3-cholamidopropyl-dimethylammonio]-1-propane-sulfonate). Protein concentrations were measured using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories). Total neprilysin activity was assayed colorimetrically, and specific activities representing an average of six independent measurements were expressed in units of pmol/μg protein/min.Western Blotting for GST, Total, and Phosphorylated Extracellular-regulated Kinase (ERK)—Subconfluent monolayers of tHBMEC, HUVEC, and LNCaP cells were lysed in radioimmune precipitation lysis buffer (50 mm Tris-HCl, pH 7.4, 1% v/v Nonidet P-40, 0.25% w/v sodium deoxycholate, 150 mm sodium chloride, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml each of aprotinin, leupeptin, pepstatin, 2 mm sodium orthovanadate) following 2-h pretreatment with 10 nm CGS24592 or vehicle and then treated for 20 min with various combinations of FGF-2, GST, or GST-FGF-2 fusion proteins, and rNEP as indicated. Lysates (50 μg each) were subjected to 10% SDS-PAGE, transferred to nitrocellulose membranes, blocked for 0.5 h, and blotted in 3% bovine serum albumin Tris-buffered saline with 0.1% Tween 20 using either anti-GST antibody (B14; 1:1000; Santa Cruz Biotechnology) or anti-phospho-ERK (197G2; 1:500; Cell Signaling Technology) as indicated for 1 h. This was followed by appropriate horseradish peroxidase-conjugated secondary antibody (sheep anti-mouse for B14 or donkey anti-rabbit for 197G2; Amersham Biosciences) at 1:4000 dilution for 0.5 h, enhanced chemoluminescence (Amersham Biosciences), and exposure to Kodak Biomax XAR film. Anti-phospho-ERK blots were stripped in 62.5 mm Tris-Cl, pH 6.8, 100 mm β-mercaptoethanol, 2% SDS, and reprobed with anti-ERK (C14; 1:1000; Santa Cruz) followed by anti-rabbit horseradish peroxidase secondary antibody. Films were scanned using an HP scanner and subjected to densitometric analysis using ImageJ and GraphPad software.RESULTSNeprilysin Cleaves FGF-2 Protein between Residues Leucine 135 and Glycine 136—To test the hypothesis that neprilysin could regulate angiogenesis in vivo, we used the murine corneal pocket assay to study the effect of rNEP on neovascularization induced by either FGF-2 or VEGF. In this assay, hydron pellets containing various concentrations of FGF-2 were implanted in the cornea ∼1 mm from the limbus and neovascularization measured 5 days later. As shown in Fig. 1, rNEP significantly inhibited FGF-2-induced neovascularization (p < 0.01) but had no effect on that of VEGF. These results suggested the possibility that FGF-2 was inactivated catalytically by neprilysin. Basic FGF is a potent proangiogenic, heparin-binding growth factor, with a primary translation product of 155 amino acids. Neprilysin hydrolyzes peptide bonds on the amino side of neutral residues; however, a protein of 155 amino acids is theoretically too large to be a substrate for neprilysin, as previous identified substrates are less than 43 amino acids (19Kenny J. Biochem. Soc. Trans. 1993; 21: 663-668Crossref PubMed Scopus (32) Google Scholar, 20Howell S. Nalbantoglu J. Crine P. Peptides. 1995; 16: 647-652Crossref PubMed Scopus (184) Google Scholar). To test whether neprilysin could hydrolyze FGF-2, we incubated rNEP with recombinant FGF-2 for 1 h and separated the products on a 14% polyacrylamide gel. Recombinant VEGF was used as control. As shown in Fig. 2A, lane 1, arrow), the molecular weight of FGF-2 protein, but not VEGF protein, was appreciably lower following rNEP incubation. The increased electrophoretic mobility of FGF-2 incubated with rNEP was blocked by the specific neprilysin inhibitor CGS24592 (21Maniara W.M. Cipriano A. Powell M.L. J. Chromatogr. B Biomed. Sci. Appl. 1998; 706: 287-294Crossref PubMed Scopus (5) Google Scholar), indicating that neprilysin and not a contaminating protease cleaved FGF-2 (Fig. 2B, lane 4). To confirm neprilysin specifically cleaves FGF-2, we performed the same digestion using either immunoprecipitated wild-type neprilysin (WT5) or enzymatically inactive neprilysin (M22) expressed using tetracycline-repressible promoter (4Sumitomo M. Milowsky M.I. Shen R. Navarro D. Dai J. Asano T. Hayakawa M. Nanus D.M. Cancer Res. 2001; 61: 3294-3298PubMed Google Scholar) and demonstrated that an intact enzyme activity is both necessary and sufficient to observe FGF-2 cleavage (Fig. 2C).FIGURE 2Neprilysin enzymatic activity cleaves FGF-2 protein. A, basic FGF or VEGF was incubated with or without rNEP for 1 h at 25 °Cin 100 mm Tris-HCl, pH 7.6, and the samples separated by 14% SDS-PAGE. Lane 1, 12.5 μm FGF-2 + 1 μm rNEP; lane 2, 12.5 μm FGF-2 alone; lane 3, 12.5 μm VEGF alone; lane 4, 12.5 μm VEGF + 1 μm rNEP. Note faster migration of FGF-2 protein treated with rNEP (arrow). B, 12.5 μm FGF-2 was incubated with or without rNEP and the neprilysin inhibitor CGS24592 for 1 h at 25°C andthe samples separated by 14% SDS-PAGE. Lane 1, Me2SO vehicle (control); lane 2, 3 μm CGS24592; lane 3, 1 μm rNEP + 12.5 μm FGF-2; lane 4, 1 μm rNEP + 12.5 μm FGF-2 + 3 μm CGS24592. Note that the addition of CGS24592 blocks the faster migration of FGF-2 protein treated with rNEP (lane 4 compared with lane 3). C, lysates from TSU-Pr1-derived WT5 and M22 cells cultured in tetracycline-free medium for 48 h containing 500 μgof total protein were subjected to immunoprecipitation with J5 antibody and incubated with 100 ng of FGF-2 for 4 h at 37 °C. Samples were analyzed by SDS-PAGE and Western blotted for both FGF-2 and neprilysin. Note the increased electrophoretic mobility seen with wild-type neprilysin (WT5), but not enzymatically deficient neprilysin (M22). D, FGF-2 (12 μm) and neprilysin (400 nm) were incubated in 50 mm Hepes, pH 7.4, 100 mm NaCl and aliquots removed at the various times (1-230 min), quenched with SDS sample buffer, and analyzed by SDS-PAGE. Coomassie-stained intact FGF-2 (upper arrow on right) was then quantified densitometrically (NIH ImageJ software) and expressed graphically as the percentage of intact FGF-2, with 100% defined as the intensity of the intact FGF-2 band at time 0, just prior to addition of rNEP. E, recombinant neprilysin was incubated with FGF-2 with (upper panel) or without (lower panel) CGS24592 and analyzed by MALDI-TOF mass spectrometry. Note the presence of a 2019-Da band, which corresponds to amino acids 136-155 of the FGF-2 protein in the absence of CGS24592. F, crystal structure of the 155-amino acid form of recombinant FGF-2 (Protein Data Bank accession code 1BFF from Ref. 23Kastrup J.S. Eriksson E.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 160-168Crossref PubMed Scopus (20) Google Scholar). The neprilysin cleavage site is highlighted. G, fibroblast growth factor-2, rNEP, and heparin were incubated for 1 h at 25°C in 100 mm Tris-HCl, pH 7.6, and subjected to 14% SDS-PAGE analysis with Coomassie Blue staining. Lane 1,1 μm rNEP alone; lane 2, 12.5 μm FGF-2 + 1 μm rNEP + 12 units/ml heparin; lane 3, 12.5 μm FGF-2 + 1 μm rNEP; lane 4, 12.5 μm FGF-2 alone. Note heparin inhibits the faster migration of FGF-2 protein treated with rNEP (lane 2). Lanes 1 and 4 are controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that FGF-2 cleavage occurs rapidly under physiologically relevant conditions, we assessed the kinetics of FGF-2 proteolysis by neprilysin, using an enzyme:substrate ratio of 1:30 and monitoring reaction progression using SDS-PAGE. As shown in Fig. 2D, 50% of FGF-2 was cleaved within the first 5-10 min, indicating rapid reaction kinetics.Next the neprilysin cleavage site on FGF-2 was localized. Fibroblast growth factor-2 and rNEP were combined with or without the neprilysin inhibitor CGS24592 and analyzed using MALDI-TOF mass spectrometry. This identified a specific 2019-Da band produced in the absence of CGS24592 that corresponded precisely to a 20-amino acid peptide located at the C terminus of the FGF-2 protein (Fig. 2E). Examination of the FGF-2 amino acid sequence confirmed the potential neprilysin cleavage site between leucine 135 and glycine 136 (22Abraham J.A. Whang J.L. Tumolo A. Mergia A. Fiddes J.C. Cold Spring Harbor Symp. Quant. Biol. 1986; 51: 657-668Crossref PubMed Google Scholar). Correlation with the three-dimensional structure of FGF-2 (23Kastrup J.S. Eriksson E.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 160-168Crossref PubMed Scopus (20) Google Scholar) indicated that the neprilysin recognition site was located at the outer edge of the FGF-2 protein, suggesting that it could fit into the neprilysin active site (Fig. 2F).Basic FGF is primarily stored in the extracellular matrix and basement membrane associated with heparan sulfate proteoglycan (HSPG). Activity of FGF-2 is controlled in part by a low affinity but high capacity interaction with HSPG. Free FGF-2 may be proteolytically degraded, as suggested by in vitro reactivity of the C-terminal portion of FGF-2 to trypsin and chymotrypsin (24Sommer A. Rifkin D.B. J. Cell Physiol. 1989; 138: 215-220Crossref PubMed Scopus (257) Google Scholar, 25Kajio T. Kawahara K. Kato K. FEBS Lett. 1992; 306: 243-246Crossref PubMed Scopus (36) Google Scholar). We hypothesized that HSPG binding could protect FGF-2 from degradation by neprilysin because leucine 135 and glycine 136 of the FGF-2 protein lie within a basic region where heparin-derived tetra- and hexasaccharides have been reported to complex with FGF-2 (26Faham S. Hileman R.E. Fromm J.R. Linhardt R.J. Rees D.C. Science. 1996; 271: 1116-1120Crossref PubMed Scopus (732) Google Scholar). Incubation of rNEP and FGF-2 plus heparin (12 units/ml) showed that heparin completely inhibited the ability of neprilysin to cleave FGF-2 (Fig. 2G, lane 2 compared with lane 3). Together, these data suggest that FGF-2 is a neprilysin substrate and that HSPG protects FGF-2 from neprilysin cleavage.Endogenous Neprilysin Expressed on Human Vascular Endothelial Cells Negatively Regulates FGF-2-induced Angiogenesis—Previous studies indicate that neprilysin is expressed by human vascular endothelial cells (9Llorens-Cortes C. Huang H. Vicart P. Gasc J.M. Paulin D. Corvol P. J. Biol. Chem. 1992; 267: 14012-14018Abstract Full Text PDF PubMed Google Scholar, 27Graf K. Koehne P. Grafe M. Zhang M. Auch-Schwelk W. Fleck E. Hypertension. 1995; 26: 230-235Crossref PubMed Scopus (71) Google Scholar). Analyses of SV40 tHBMEC (12Schweitzer K.M. Vicart P. Delouis C. Paulin D. Drager A.M. Langenhuijsen M.M. Weksler B.B. Lab. Investig. 1997; 76: 25-36PubMed Google Scholar) and HUVEC revealed neprilysin enzyme-specific activities of 197 pmol/μg/min and 36 pmol/μg/min, respectively (data not shown). To assess the functional effect of endogenous neprilysin on FGF-2-induced angiogenesis in vitro, we used a capillary array formation assay to measure the effects of FGF-2 with and without the neprilysin inhibitor CGS24592 in tHBMEC cells plated on Matrigel-coated plates. As shown in Fig. 3A, cells grown in the presence of FGF-2 and CGS24592 demonstrated significantly more arrays compared with cells grown in FGF-2 alone, CGS24592 alone, or the untreated negative control (p < 0.05). We next assessed endothelial cell growth in tHBMEC treated with 600 pg/ml FGF-2 and with increasing concentrations of CGS24592. Transduced HBMEC incorporated more MTT as a function of CGS24592 concentration, suggesting that inhibition of endogenous neprilysin activity results in increased FGF-2-induced cell growth (p < 0.03, Fig. 3B). Similar results were obtained for HUVEC cells (data not shown). These data show that neprilysin expressed on vascular endothelial cells regulates FGF-2-induced angiogenesis.FIGURE 3Endogenous neprilysin inhibition enhances FGF-2-induced angiogenesis. A, transduced human bone marrow microvascular endothelial cells were assayed using a Matrigel capillary array formation assay. Cells were plated on growth factor-reduced Matrigel in the presence (+) or absence (-) of CGS24592 (30 nm) for 2 h followed by the addition of FGF-2 (5 ng/ml) for 4 h as indicated. Representative photographs were then taken. B, transduced human bone marrow microvascular en" @default.
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• W2004865667 date "2006-11-01" @default.
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