The 166-amino acid form of vascular endothelial growth factor (VEGF,,) is a mitogen for vascular endothelial cells and a potent angiogenic factor.Expression of a chimeric receptor containing the extracellular domain of the f l k -l receptor fused to the transmembrane and intracellular domains of the human c-fma receptor in NIH-3T3 cells, resulted in the appearance of high affinity binding sites for laaI-VEGF,, on transfected cells.The binding of ' 261-VEGF1, to the flk-llfma chimeric receptor of the transfected cells as well as the VEGF,,induced autophosphorylation of the chimeric receptors were inhibited in the presence of low concentrations of heparin (1-10 pg/ml).In contrast, similar concentrations of heparin potentiated the binding of laaI-VEGF,, to the endogenous VEGF receptors of the transfected cells, indicating that to some extent, the effect of heparin on 'aaI-VEGF,, binding is receptor type-dependent.A soluble fusion protein containing the extracellular domain of flk-1 fused to alkaline phosphatase ( f l k -l l SEW) was used to study the effects of heparin on the binding of ' 261-VEGF,, to flk-1 in a cell-free environment.The fusion protein specifically inhibited VEGF,,induced proliferation of vascular endothelial cells, but bound laaI-VEGF,, inefficiently in the absence of heparin.Addition of low concentrations of heparin or heparan sulfate (0.1-1 pg/ml) resulted in a strong potentiation of 'aaI-VEGF,, binding, whereas higher heparin or heparan sulfate concentrations inhibited the binding.The effect of heparin on the binding of 'aaI-VEGFl, to flk-IISEAP could not be mimicked by desulfated heparin or by chondroitin sulfate.Both bFGF and aFGF inhibited the binding when low concentrations of heparin were added to the binding reaction.However, higher concentrations of heparin abolished the inhibition, indicating that the inhibition is probably caused by competition for available heparin.Taken as a whole, these results indicate that heparin-like molecules regulate the binding of VEGF,, to its receptors in complex ways which depend on the heparin binding properties of VEGF,,, on the specific VEGF receptor type involved, and on the amount and composition of heparin-like molecules that are present on the cell surface of VEGF receptor containing cells.
Abstract Objective To study the efficacy and safety of phosphatidylserine (PS) containing Omega3 long-chain polyunsaturated fatty acids attached to its backbone (PS-Omega3) in reducing attention-deficit/ hyperactivity disorder (ADHD) symptoms in children. Method A 15-week, double-blind, placebo-controlled phase followed by an open-label extension of additional 15 weeks. Two hundred ADHD children were randomized to receive either PS-Omega3 or placebo, out of them, 150 children continued into the extension. Efficacy was assessed using Conners’ parent and teacher rating scales (CRS-P,T), Strengths and Difficulties Questionnaire (SDQ), and Child Health Questionnaire (CHQ). Safety evaluation included adverse events monitoring. Results The key finding of the double-blind phase was the significant reduction in the Global:Restless/impulsive subscale of CRS-P and the significant improvement in Parent impact-emotional (PE) subscale of the CHQ, both in the PS-Omega3 group. Exploratory subgroup analysis of children with a more pronounced hyperactive/impulsive behavior, as well as mood and behavior-dysregulation, revealed a significant reduction in the ADHD-Index and hyperactive components. Data from the open-label extension indicated sustained efficacy for children who continued to receive PS-Omega3. Children that switched to PS-Omega3 treatment from placebo showed a significant reduction in subscales scores of both CRS-P and the CRS-T, as compare to baseline scores. The treatment was well tolerated. Conclusions The results of this 30-week study suggest that PS-Omega3 may reduce ADHD symptoms in children. Preliminary analysis suggests that this treatment may be especially effective in a subgroup of hyperactive-impulsive, emotionally and behaviorally-dysregulated ADHD children.
Introduction: Seeding polymer-based synthetic vascular grafts with endothelial cells (EC) improves grafts biocompatibility and consequently grafts patency. EC adhesion and survival on the synthetic polymer remains a considerable challenge. We used pseudo-typed retroviral vectors to modify EC to co-express fibulin-5 and VEGF 165 and created a unique EC phenotype with increased adhesion and enhanced proliferation and used these cells to seed vascular grafts. Methods: Polymer-based grafts seeded with autologous venous EC transduced to co-express fibulin-5 and VEGF 165 were implanted in the carotid arteries of the donor sheep. 18 grafts (8 seeded grafts with EC co-expressing fibulin-5 and VEGF, 6 seeded with control EC and 4 bare grafts) were implanted for two week in nine sheep. Six months studies were performed on 18 grafts (6 study, 6 seeded with control EC and 6 bare) implanted in 18 sheep. Histological studies for cell adhesion, intra-luminal thrombosis and transgene expression were tested in implanted grafts after angiography. Results: At two weeks all 8 grafts seeded with EC co-expressing fibulin-5 and VEGF were patent with no significant thrombosis and 73% coverage by seeded EC, while 3/10 control grafts were thrombosed and only 30% of seeded unmodified EC remained on grafts luminal surface. At 6 months, 5/6 grafts seeded with EC co-expressing fibulin-5 and VEGF were patent while 9/12 control grafts (6 bare, 6 seeded with unmodified EC, p<0.04) were occluded. At 6 months, the genetically modified cells were identified on the graft surface and these grafts had improved EC coverage and reduced intra-luminal neo-intima and thrombosis. No local or systemic side effects were attributed to the implanted grafts. Conclusion : Polymer-based vascular grafts seeded with EC transduced to co-express fibulin-5 and VEGF have lower propensity to thrombosis and improved patency. Use of grafts seeded with EC co-expressing fibulin-5 and VEGF provides a potentially clinically relevant method for small caliber blood vessel engineering.
Abstract Background Phosphatidylserine (PS) is a naturally occurring phospholipid present in the inner leaflet of mammalian plasma membranes. Administration of PS extracted from bovine cortex (BC-PS), which contains high levels of omega-3 long chain polyunsaturated fatty acid (LC-PUFA) attached to its backbone, resulted in positive effects on brain functions such as learning and memory. Recently, a novel marine-sourced PS with omega-3 LC-PUFA attached to its backbone was developed (PS-DHA). In the present study, we evaluated the safety profile of the novel PS preparation in non-demented elderly with memory complaints. The efficacy study of this novel formulation indicated that PS-DHA may ameliorate cognitive deficits in non-demented elderly population. Methods 157 non-demented elderly participants with memory complaints were randomized to receive either PS-DHA (300 mg PS/day) or placebo for 15 weeks. Standard biochemical and hematological safety parameters, blood pressure and heart rate were evaluated at baseline and endpoint. 122 participants continued into an open-label extension for additional 15 weeks, in which they all consumed PS-DHA (100 mg PS/day) and were evaluated for their blood pressure, heart rate and weight at endpoint. Adverse events were monitored throughout the double-blind and open-label phases. Results 131 participants completed the double-blind phase. No significant differences were found in any of the tested safety parameters between the study groups, or within each group. 121 participants completed the open-label phase. At the end of this phase, there was a reduction in resting diastolic blood pressure and a slight weight gain among participants who consumed PS-DHA for 30 weeks. Conclusions The results of this study indicate that consumption of PS-DHA at a dosage of 300 mg PS/day for 15 weeks, or 100 mg PS/day for 30 weeks, is safe, well tolerated, and does not produce any negative effects in the tested parameters. Trial registration clinicaltrials. gov, identifier: NCT00437983
The products of the neuropilin-1 (Np-1) and neuropilin-2 (Np-2) genes are receptors for factors belonging to the class 3 semaphorin family and participate in the guidance of growing axons to their targets. In the presence of heparin-like molecules, both receptors also function as receptors for the heparin-binding 165-amino acid isoform of vascular endothelial growth factor (VEGF165). Both receptors are unable to bind to the 121-amino acid isoform of vascular endothelial growth factor (VEGF121), which lacks a heparin-binding domain. Interestingly, complexes corresponding in size to125I-VEGF121·neuropilin complexes are formed when 125I-VEGF121 is bound and cross-linked to porcine aortic endothelial cells co-expressing VEGFR-1 and either Np-1 or Np-2. These complexes do not seem to represent complexes of 125I-VEGF121 with a truncated form of VEGFR-1, presumably formed as a result of the presence of Np-1 or Np-2 in the cells, because such truncated forms could not be detected with anti-VEGFR-1 antibodies. Antibodies directed against VEGFR-1 co-immunoprecipitated the125I-VEGF121·Np-2 sized cross-linked complex along with 125I-VEGF121·VEGFR-1 complexes from cells expressing both VEGFR-1 and Np-2 but not from control cells, indicating that VEGFR-1 and Np-2 associate with each other. To perform the reciprocal experiment we have expressed in porcine aortic endothelial cells a Np-2 receptor containing an in-framemyc epitope at the C terminus. Surprisingly, themyc-tagged Np-2 receptor lost most of its VEGF165 binding capacity but not its semaphorin-3F binding ability. Nevertheless, when Np-2myc was co-expressed in cells with VEGFR-1, it partially regained its VEGF165binding ability. Antibodies directed against the mycepitope co-immunoprecipitated125I-VEGF165·Np-2myc and125I- VEGF165·VEGFR-1 complexes from cells co-expressing VEGFR-1 and Np-2myc, indicating again that VEGFR-1 associates with Np-2. Our experiments therefore indicate that Np-2, and possibly also Np-1, associate with VEGFR-1 and that such complexes may be part of a cell membrane-associated signaling complex. The products of the neuropilin-1 (Np-1) and neuropilin-2 (Np-2) genes are receptors for factors belonging to the class 3 semaphorin family and participate in the guidance of growing axons to their targets. In the presence of heparin-like molecules, both receptors also function as receptors for the heparin-binding 165-amino acid isoform of vascular endothelial growth factor (VEGF165). Both receptors are unable to bind to the 121-amino acid isoform of vascular endothelial growth factor (VEGF121), which lacks a heparin-binding domain. Interestingly, complexes corresponding in size to125I-VEGF121·neuropilin complexes are formed when 125I-VEGF121 is bound and cross-linked to porcine aortic endothelial cells co-expressing VEGFR-1 and either Np-1 or Np-2. These complexes do not seem to represent complexes of 125I-VEGF121 with a truncated form of VEGFR-1, presumably formed as a result of the presence of Np-1 or Np-2 in the cells, because such truncated forms could not be detected with anti-VEGFR-1 antibodies. Antibodies directed against VEGFR-1 co-immunoprecipitated the125I-VEGF121·Np-2 sized cross-linked complex along with 125I-VEGF121·VEGFR-1 complexes from cells expressing both VEGFR-1 and Np-2 but not from control cells, indicating that VEGFR-1 and Np-2 associate with each other. To perform the reciprocal experiment we have expressed in porcine aortic endothelial cells a Np-2 receptor containing an in-framemyc epitope at the C terminus. Surprisingly, themyc-tagged Np-2 receptor lost most of its VEGF165 binding capacity but not its semaphorin-3F binding ability. Nevertheless, when Np-2myc was co-expressed in cells with VEGFR-1, it partially regained its VEGF165binding ability. Antibodies directed against the mycepitope co-immunoprecipitated125I-VEGF165·Np-2myc and125I- VEGF165·VEGFR-1 complexes from cells co-expressing VEGFR-1 and Np-2myc, indicating again that VEGFR-1 associates with Np-2. Our experiments therefore indicate that Np-2, and possibly also Np-1, associate with VEGFR-1 and that such complexes may be part of a cell membrane-associated signaling complex. vascular endothelial growth factor bis(sulfosuccinimidyl) suberate vascular endothelial growth factor receptor-1 vascular endothelial growth factor receptor-2 polyacrylamide gel electrophoresis 165-amino acid form of vascular endothelial growth factor 121-amino acid form of vascular endothelial growth factor neuropilin-1 neuropilin-2 porcine aortic endothelial cells semaphorin placenta growth factor phosphate-buffered saline alkaline phosphatase. The various forms of the growth factors belonging to the VEGF1 family (VEGF, PlGF, VEGF-B, VEGF-C, and VEGF-D) act as inducers and modulators of angiogenesis in vivo (1Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3129) Google Scholar, 2Eriksson U. Alitalo K. Curr. Top. Microbiol. Immunol. 1999; 237: 41-57Crossref PubMed Scopus (151) Google Scholar, 3Persico M.G. Vincenti V. Dipalma T. Curr. Top. Microbiol. Immunol. 1999; 237: 31-40Crossref PubMed Scopus (145) Google Scholar). The active forms of the VEGF family members are synthesized as homodimers (4Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4414) Google Scholar, 5Keck P.J. Hauser S.D. Krivi G. Sanzo K. Warren T. Feder J. Connolly D.T. Science. 1989; 246: 1309-1312Crossref PubMed Scopus (1794) Google Scholar) or as heterodimers with other VEGF family members such as PlGF (6DiSalvo J. Bayne M.L. Conn G. Kwok P.W. Trivedi P.G. Soderman D.D. Palisi T.M. Sullivan K.A. Thomas K.A. J. Biol. Chem. 1995; 270: 7717-7723Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Targeted disruption of the VEGF gene has shown that angiogenesis is severely disrupted even in heterozygous animals containing a single functional allele of the VEGF gene. It is therefore believed that the maintenance of correct levels of VEGF in vivo is critical for the development of the cardiovascular system (7Ferrara N. Carvermoore K. Chen H. Dowd M. Lu L. Oshea K.S. Powellbraxton L. Hillan K.J. Moore M.W. Nature. 1996; 380: 439-442Crossref PubMed Scopus (3021) Google Scholar, 8Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3427) Google Scholar). Five splice forms of human VEGF ranging from 121 to 206 amino acids (VEGF121–VEGF206) have been characterized (4Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4414) Google Scholar, 5Keck P.J. Hauser S.D. Krivi G. Sanzo K. Warren T. Feder J. Connolly D.T. Science. 1989; 246: 1309-1312Crossref PubMed Scopus (1794) Google Scholar, 9Poltorak Z. Cohen T. Sivan R. Kandelis Y. Spira G. Vlodavsky I. Keshet E. Neufeld G. J. Biol. Chem. 1997; 272: 7151-7158Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 10Houck K.A. Ferrara N. Winer J. Cachianes G. Li B. Leung D.W. Mol. Endocrinol. 1991; 5: 1806-1814Crossref PubMed Scopus (1230) Google Scholar). These differ primarily in the presence or the absence of the heparin-binding domains encoded by exons 6 and 7, giving rise to forms that differ in their heparin and heparan-sulfate binding ability (11Neufeld G. Cohen T. Gitay-Goren H. Poltorak Z. Tessler S. Gengrinovitch S. Levi B.-Z. Cancer Metastasis Rev. 1996; 15: 153-158Crossref PubMed Scopus (145) Google Scholar). Likewise, other VEGF family members such as PlGF and VEGF-B are also expressed in several forms that differ in their heparin binding ability. For example, the peptide encoded by exon 6 of PlGF is found only in PlGF-2 and confers a heparin binding ability to PLGF-2, whereas PlGF-1 does not bind to heparin (12Maglione D. Guerriero V. Viglietto G. Ferraro M.G. Aprelikova O. Alitalo K. Delvecchio S. Lei K.J. Chou J.Y. Persico M.G. Oncogene. 1993; 8: 925-931PubMed Google Scholar).All VEGF isoforms bind to the tyrosine-kinase receptors VEGFR-1 (flt-1) (13Shibuya M. Yamaguchi S. Yamane A. Ikeda T. Tojo A. Matsushime H. Sato M. Oncogene. 1990; 5: 519-524PubMed Google Scholar) and VEGFR-2 (KDR/flk-1) (14Terman B.I. Carrion M.E. Kovacs E. Rasmussen B.A. Eddy R.L. Shows T.B. Oncogene. 1991; 6: 1677-1683PubMed Google Scholar). The binding of VEGF to VEGFR-2 initiates intracellular signal transduction (1Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3129) Google Scholar, 15Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Bôhlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1389) Google Scholar, 16Devries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1880) Google Scholar, 17Yamane A. Seetharam L. Yamaguchi S. Gotoh N. Takahashi T. Neufeld G. Shibuya M. Oncogene. 1994; 9: 2683-2690PubMed Google Scholar, 18Millauer B. Wizigmannvoos S. Schnurch H. Martinez R. Moller N.P.H. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1752) Google Scholar) and is correlated with the induction of endothelial cell proliferation, migration, and in vivo angiogenesis (19Millauer B. Shawver L.K. Plate K.H. Risau W. Ullrich A. Nature. 1994; 367: 576-579Crossref PubMed Scopus (1158) Google Scholar, 20Waltenberger J. Claessonwelsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). By contrast, the activation of VEGFR-1 does not seem to result in the induction of cell proliferation or angiogenesis, although exceptions have been observed (21Ziche M. Maglione D. Ribatti D. Morbidelli L. Lago C.T. Battisti M. Paoletti I. Barra A. Tucci M. Parise G. Vincenti V. Granger H.J. Viglietto G. Persico M.G. Lab. Invest. 1997; 76: 517-531PubMed Google Scholar, 22Landgren E. Schiller P. Cao Y. Claesson-Welsh L. Oncogene. 1998; 16: 359-367Crossref PubMed Scopus (147) Google Scholar). However, the activation of VEGFR-1 seems to enhance cell migration (20Waltenberger J. Claessonwelsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 23Seetharam L. Gotoh N. Maru Y. Neufeld G. Yamaguchi S. Shibuya M. Oncogene. 1995; 10: 135-147PubMed Google Scholar, 24Clauss M. Weich H. Breier G. Knies U. Roeckl W. Waltenberger J. Risau W. J. Biol. Chem. 1996; 271: 17629-17634Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar, 25Barleon B. Sozzani S. Zhou D. Weich H.A. Mantovani A. Marme D. Blood. 1996; 87: 3336-3343Crossref PubMed Google Scholar). Both receptors play pivotal roles in embryonic vasculogenesis and angiogenesis. Embryos lacking the VEGFR-2 gene die before birth because differentiation of endothelial cells does not take place and blood vessels do not form (26Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3332) Google Scholar). In contrast, disruption of the gene encoding the VEGFR-1 receptor did not prevent the differentiation of endothelial cells in homozygous animals, but the development of functional blood vessels from these endothelial cells was severely impaired (27Fong G.H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2202) Google Scholar).Endothelial cells also contain another type of VEGF receptor possessing a lower mass than either VEGFR-2 or VEGFR-1 (28Vaisman N. Gospodarowicz D. Neufeld G. J. Biol. Chem. 1990; 265: 19461-19466Abstract Full Text PDF PubMed Google Scholar, 29Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar). It was subsequently found that these smaller VEGF receptors of the endothelial cells are isoform-specific receptors that bind VEGF165 but not VEGF121 (30Gitay-Goren H. Cohen T. Tessler S. Soker S. Gengrinovitch S. Rockwell P. Klagsbrun M. Levi B.-Z. Neufeld G. J. Biol. Chem. 1996; 271: 5519-5523Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Additional experiments revealed several types of prostate and breast cancer-derived cell lines that express unusually large amounts of these isoform-specific receptors (31Soker S. Fidder H. Neufeld G. Klagsbrun M. J. Biol. Chem. 1996; 271: 5761-5767Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). The receptors were purified from such cells using affinity chromatography on VEGF165 affinity matrices followed by partial protein sequencing and were found to be the products of the neuropilin-1 (Np-1) gene (32Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar). It was subsequently observed that the heparin-binding form of placenta growth factor (PlGF-2) and VEGF-B, two additional members of the VEGF family of growth factors, are also able to bind to Np-1 (33Migdal M. Huppertz B. Tessler S. Comforti A. Shibuya M. Reich R. Baumann H. Neufeld G. J. Biol. Chem. 1998; 273: 22272-22278Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 34Makinen T. Olofsson B. Karpanen T. Hellman U. Soker S. Klagsbrun M. Eriksson U. Alitalo K. J. Biol. Chem. 1999; 274: 21217-21222Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). When the role of Np-1 as a VEGF receptor was discovered, it was already known that Np-1 functions in the nervous system as receptor for sema-3A. sema-3A is a Np-1 agonist that causes repulsion of growing tips of axons (35He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 36Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar). It was recently observed that sema-3A is also able to inhibit migration of endothelial cells (37Miao H.Q. Soker S. Feiner L. Alonso J.L. Raper J.A. Klagsbrun M. J. Cell Biol. 1999; 146: 233-242Crossref PubMed Scopus (435) Google Scholar). These results indicate that signaling via Np-1 affects angiogenesis and possibly the development of the cardiovascular system. Targeted disruption of the Np-1 gene resulted in severe cardiovascular defects, confirming these suspicions (38Kawasaki T. Kitsukawa T. Bekku Y. Matsuda Y. Sanbo M. Yagi T. Fujisawa H. Development. 1999; 126: 4895-4902Crossref PubMed Google Scholar, 39Kitsukawa T. Shimizu M. Sanbo M. Hirata T. Taniguchi M. Bekku Y. Yagi T. Fujisawa H. Neuron. 1997; 19: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 40Kitsukawa T. Shimono A. Kawakami A. Kondoh H. Fujisawa H. Development. 1995; 121: 4309-4318Crossref PubMed Google Scholar). Np-1 is part of a gene family that includes the closely related receptor Np-2. In the nervous system Np-2 is activated by another class 3 semaphorin, sema-3F, which also induces the repulsion of axons that express Np-2 (41Chen H. Chedotal A. He Z. Goodman C.S. Tessier-Lavigne M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). We have recently observed that Np-2 is also able to bind VEGF165 and PlGF-2 but not VEGF121. However, unlike Np-1, Np-2 was also able to interact with the VEGF145 form of VEGF. VEGF145lacks the peptide encoded by exon 7 of VEGF, which is included in VEGF165, but contains instead the heparin-binding domain encoded by exon 6 of the VEGF gene (9Poltorak Z. Cohen T. Sivan R. Kandelis Y. Spira G. Vlodavsky I. Keshet E. Neufeld G. J. Biol. Chem. 1997; 272: 7151-7158Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 42Gluzman-Poltorak Z. Cohen T. Herzog Y. Neufeld G. J. Biol. Chem. 2000; 275: 18040-18045Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar).The neuropilins have a short intracellular domain and are unlikely to function as independent receptors. Indeed, no responses to VEGF165 were observed when cells expressing either Np-1 or Np-2 but no other VEGF receptors were stimulated with VEGF165 (32Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar, 42Gluzman-Poltorak Z. Cohen T. Herzog Y. Neufeld G. J. Biol. Chem. 2000; 275: 18040-18045Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). It was recently found that plexins form complexes with neuropilins and that these complexes mediate signal transduction by sema-3A (43Takahashi T. Fournier A. Nakamura F. Wang L.H. Murakami Y. Kalb R.G. Fujisawa H. Strittmatter S.M. Cell. 1999; 99: 59-69Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 44Tamagnone L. Artigiani S. Chen H. He Z. Ming G.I. Song H. Chedotal A. Winberg M.L. Goodman C.S. Poo M. Tessier-Lavigne M. Comoglio P.M. Cell. 1999; 99: 71-80Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar). It is possible that neuropilins associate with additional cell surface molecules to form complexes that transduce VEGF signaling. We present evidence indicating that Np-2 and possibly also Np-1 form complexes with the VEGFR-1 receptor and that the formation of these complexes changes the binding characteristics of neuropilins so that they are now able to bind VEGF121, a splice form that is not recognized by neuropilins in cells that do not express VEGFR-1.DISCUSSIONNp-1 and Np-2 were originally found to function as receptors for several class 3 semaphorins that repel growing tips of axons during the development of the nervous system (35He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar, 36Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (994) Google Scholar, 41Chen H. Chedotal A. He Z. Goodman C.S. Tessier-Lavigne M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). These discoveries were followed by experiments that have demonstrated that Np-1 and Np-2 can function in addition as receptors for VEGF165, one of the heparin-binding forms of the angiogenic factor VEGF (32Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar, 42Gluzman-Poltorak Z. Cohen T. Herzog Y. Neufeld G. J. Biol. Chem. 2000; 275: 18040-18045Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). These experiments indicated that the neuropilins may play a role in cardiovascular biology, in addition to their role in the nervous system. In the case of Np-1 these expectations were verified when it was shown that targeted disruption of the Np-1 gene results in severe cardiovascular defects (38Kawasaki T. Kitsukawa T. Bekku Y. Matsuda Y. Sanbo M. Yagi T. Fujisawa H. Development. 1999; 126: 4895-4902Crossref PubMed Google Scholar). In agreement with this observation it was found that the Np-1 agonist sema-3A inhibits migration of endothelial cells (37Miao H.Q. Soker S. Feiner L. Alonso J.L. Raper J.A. Klagsbrun M. J. Cell Biol. 1999; 146: 233-242Crossref PubMed Scopus (435) Google Scholar), but the consequences of the binding of VEGF to Np-1 are not completely clear as yet. In contrast, mice lacking functional Np-2 receptors are viable, and no vascular defects were reported so far (51Giger R.J. Cloutier J.F. Sahay A. Prinjha R.K. Levengood D.V. Moore S.E. Pickering S. Simmons D. Rastan S. Walsh F.S. Kolodkin A.L. Ginty D.D. Geppert M. Neuron. 2000; 25: 29-41Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar,52Chen H. Bagri A. Zupicich J.A. Zou Y. Stoeckli E. Pleasure S.J. Lowenstein D.H. Skarnes W.C. Chedotal A. Tessier-Lavigne M. Neuron. 2000; 25: 43-56Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Nevertheless, the absence of vascular defects in these gene targeted mice does not necessarily preclude a physiological role for these receptors in vascular biology because the absence of a phenotype may be explained by redundancy with other signaling pathways.We have not been able to demonstrate any biological responses to VEGF165 in PAE cells expressing either recombinant Np-1 or recombinant Np-2 receptors and no other types of VEGF receptors (32Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar,42Gluzman-Poltorak Z. Cohen T. Herzog Y. Neufeld G. J. Biol. Chem. 2000; 275: 18040-18045Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). These observations suggested that for the transduction of VEGF signals the neuropilins may perhaps have to associate with other membrane-bound proteins. Neuropilins possess short intracellular domains, and it was demonstrated that binding of sema-3A to Np-1 is not sufficient for induction of sema-3A mediated growth cone collapse (53Nakamura F. Tanaka M. Takahashi T. Kalb R.G. Strittmatter S.M. Neuron. 1998; 21: 1093-1100Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). It was indeed found that neuropilins form complexes with plexin receptors to be able to transduce semaphorin signals (43Takahashi T. Fournier A. Nakamura F. Wang L.H. Murakami Y. Kalb R.G. Fujisawa H. Strittmatter S.M. Cell. 1999; 99: 59-69Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 44Tamagnone L. Artigiani S. Chen H. He Z. Ming G.I. Song H. Chedotal A. Winberg M.L. Goodman C.S. Poo M. Tessier-Lavigne M. Comoglio P.M. Cell. 1999; 99: 71-80Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar). Our binding/cross-linking experiments and co-immunoprecipitation experiments indicate that in addition, Np-2 can form complexes with VEGFR-1. Our experiments also suggest that Np-1 too can associate with VEGFR-1. This was recently verified in a manuscript that was published during the preparation of this manuscript in which complexes between Np-1 and VEGFR-1 were observed using completely different methods (49Fuh G. Garcia K.C. De Vos A.M. J. Biol. Chem. 2000; 275: 26690-26695Abstract Full Text Full Text PDF PubMed Google Scholar).The mechanism by which VEGFR-1 enables the binding of VEGF121 to Np-1 and Np-2 is unclear. The binding of VEGFR-1 to the neuropilins may induce a neuropilin conformation that binds VEGF121 with increased affinity. It is possible that VEGF121 binds initially to VEGFR-1, placing the bound VEGF121 in close proximity to neuropilins in cells co-expressing both receptor types and effectively increasing the affinity of the neuropilins toward VEGF121. The effect of VEGFR-1 on VEGF121 binding may therefore be similar to the potentiating effect that heparin-like molecules have on the binding of VEGF165 to neuropilins (32Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar, 33Migdal M. Huppertz B. Tessler S. Comforti A. Shibuya M. Reich R. Baumann H. Neufeld G. J. Biol. Chem. 1998; 273: 22272-22278Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 42Gluzman-Poltorak Z. Cohen T. Herzog Y. Neufeld G. J. Biol. Chem. 2000; 275: 18040-18045Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar).VEGFR-1 and Np-2 may be able to form complexes prior to the addition of VEGF. Alternatively, it is possible that VEGFR-1 binds to Np-2 only subsequent to the binding of VEGF to VEGFR-1. We have not been able to differentiate between these two possibilities. We have attempted to detect co-immunoprecipitated Np-2 or VEGFR-1 using Western blot analysis, but we have failed regardless of whether the cells were exposed or not to VEGF prior to the immunoprecipitation. It is possible that these experiments failed because the sensitivity of the assays was insufficient or because the VEGFR-1·Np-2 complexes are sensitive to the detergents used during the solubilization of the cells, making the detection of VEGFR-1·Np-2 complexes by less sensitive techniques than the technique we have used difficult. To circumvent these problems we have therefore used antibodies to immunoprecipitate recombinant receptors that have been covalently cross-linked to125I-VEGF prior to the immunoprecipitation using a previously described method (50Omura T. Miyazawa K. Ostman A. Heldin C.H. J. Biol. Chem. 1997; 272: 23317-23322Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The method we used utilized the high sensitivity afforded by the use of 125I but did not allow us to determine whether complex formation between Np-2 and VEGFR-1 was VEGF-dependent or not.We have no data regarding the biological significance of VEGFR-1·Np-2 complexes at this stage. The formation of complexes between Np-2 and VEGFR-1 may contribute to VEGF-induced signal transduction by VEGFR-1. If that assumption is correct, then it may provide a clue to a puzzling observation. Mice deficient in VEGFR-1 expression die before birth because of severe cardiovascular defects (27Fong G.H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2202) Google Scholar). In contrast, mice that retain the extracellular and trans-membrane domains of VEGFR-1 but lack the signaling tyrosine-kinase domain develop normally (54Hiratsuka S. Minowa O. Kuno J. Noda T. Shibuya M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9349-9354Crossref PubMed Scopus (884) Google Scholar). It is unclear how the extracellular domain of VEGFR-1 is able to restore the normal embryonic development of mice. It is possible that the extracellular domain is required for VEGF sequestration, so as to limit the activity of VEGF. Alternatively, the extracellular domain may associate with another membrane protein to form a signaling holo-receptor. It is possible that Np-2 and Np-1 may perhaps participate in the formation of such a putative VEGFR-1 containing holo-receptor.In the course of our experiments we have found that when amyc epitope is inserted in-frame after the conserved SEA terminal tripeptide of Np-2a, the modified Np-2myc receptor loses most of its VEGF165binding ability. It was shown that Np-1 and Np-2 can form homodimers and heterodimers (55Chen H. He Z. Bagri A. Tessier-Lavigne M. Neuron. 1998; 21: 1283-1290Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Formation of such dimers may perhaps be required for high affinity binding of VEGF to neuropilins. The insertion of themyc epitope may perhaps interfere with dimer formation and consequently with VEGF binding. Interestingly, the VEGF165binding ability of Np-2myc was restored to some extent in cells co-expressing VEGFR-1, perhaps because following complex formation a high affinity VEGF-binding conformation of Np-2myc is favored. Interestingly, the sema-3F binding properties of Np-2 were not affected by the introduction of themyc epitope, perhaps because the sema-3F-binding domain of Np-2 seems to be distinct from the VEGF-binding domain as suggested by the results of the competition experiments.To conclude, our experiments indicate that VEGFR-1 forms complexes with Np-2 and possibly also with Np-1. The presence of VEGFR-1 changes the specificity of VEGF binding, allowing VEGF121 to bind to Np-2. However, the biological function of these VEGFR-1·Np-2 complexes is still unclear. In addition our experiments indicate that changes in the intracellular domain of Np-2 can affect VEGF binding to Np-2 and provide evidence indicating that VEGF and semaphorins bind to nonoverlapping sites in the extracellular part of Np-2. The various forms of the growth factors belonging to the VEGF1 family (VEGF, PlGF, VEGF-B, VEGF-C, and VEGF-D) act as inducers and modulators of angiogenesis in vivo (1Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3129) Google Scholar, 2Eriksson U. Alitalo K. Curr. Top. Microbiol. Immunol. 1999; 237: 41-57Crossref PubMed Scopus (151) Google Scholar, 3Persico M.G. Vincenti V. Dipalma T. Curr. Top. Microbiol. Immunol. 1999; 237: 31-40Crossref PubMed Scopus (145) Google Scholar). The active forms of the VEGF family members are synthesized as homodimers (4Leung D.W. Cachianes G. Kuang W.J. Goeddel D.V. Ferrara N. Science. 1989; 246: 1306-1309Crossref PubMed Scopus (4414) Google Scholar, 5Keck P.J. 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The present study is an open-label extension (OLE) aimed at evaluating the effect of 100 mg/day of phosphatidylserine enriched with docosahexaenoic acid (PS-DHA) on cognitive performance in nondemented elderly individuals with memory complaints.From the participants who completed the core study, 122 continued with a 15-week OLE. Efficacy was assessed using a computerized tool and the Clinical Global Impression of Change (CGI-C) rating scale.A significant improvement in sustained attention and memory recognition was observed in the PS-DHA naïve group, while the PS-DHA continuers maintained their cognitive status. Additionally, a significant improvement in CGI-C was observed in the naïve group.The results demonstrate that consumption of 100 mg/day of PS-DHA might be associated with improving or maintaining cognitive status in elderly subjects with memory complaints.
