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W2061004014 abstract "Expression of dystroglycan (DG) by cultured bovine aortic endothelial (BAE) cells was confirmed by cDNA cloning from a BAE cDNA library, Northern blotting of mRNA, Western blotting of membrane proteins, and double immunostaining with antibodies against βDG and platelet endothelial cell adhesion molecule-1. Immunocytochemical analysis revealed localization of DG in multiple plaques on the basal side of resting cells. This patchy distribution was obscured in migrating cells, in which the most prominent staining was observed in the trailing edge anchoring the cells to the substratum. Biotin-labeled laminin-1 overlay assay of dissociated BAE membrane proteins indicated the interaction of laminin-1 with αDG. The laminin α5 globular domain fragment expressed in bacteria and labeled with biotin could also bind αDG on the membrane blot, and the unlabeled fragment disrupted the binding of biotin-laminin-1 to αDG. The interaction of biotin-laminin-1 with αDG was inhibited by soluble αDG contained in the conditioned medium from DG cDNA-transfected BAE cells and by a series of glycosaminoglycans (heparin, dextran sulfate, and fucoidan). Soluble αDG in the conditioned medium inhibited the adhesion of BAE cells to laminin-1-coated dishes, whereas it had no effect on their adhesion to fibronectin. All three glycosaminoglycans that disrupted the biotin-laminin-1 binding to αDG inhibited BAE cell adhesion to laminin-1, whereas they failed to inhibit the adhesion to fibronectin. These results indicate a role of DG as a non-integrin laminin receptor involved in vascular endothelial cell adhesion to the extracellular matrix. Expression of dystroglycan (DG) by cultured bovine aortic endothelial (BAE) cells was confirmed by cDNA cloning from a BAE cDNA library, Northern blotting of mRNA, Western blotting of membrane proteins, and double immunostaining with antibodies against βDG and platelet endothelial cell adhesion molecule-1. Immunocytochemical analysis revealed localization of DG in multiple plaques on the basal side of resting cells. This patchy distribution was obscured in migrating cells, in which the most prominent staining was observed in the trailing edge anchoring the cells to the substratum. Biotin-labeled laminin-1 overlay assay of dissociated BAE membrane proteins indicated the interaction of laminin-1 with αDG. The laminin α5 globular domain fragment expressed in bacteria and labeled with biotin could also bind αDG on the membrane blot, and the unlabeled fragment disrupted the binding of biotin-laminin-1 to αDG. The interaction of biotin-laminin-1 with αDG was inhibited by soluble αDG contained in the conditioned medium from DG cDNA-transfected BAE cells and by a series of glycosaminoglycans (heparin, dextran sulfate, and fucoidan). Soluble αDG in the conditioned medium inhibited the adhesion of BAE cells to laminin-1-coated dishes, whereas it had no effect on their adhesion to fibronectin. All three glycosaminoglycans that disrupted the biotin-laminin-1 binding to αDG inhibited BAE cell adhesion to laminin-1, whereas they failed to inhibit the adhesion to fibronectin. These results indicate a role of DG as a non-integrin laminin receptor involved in vascular endothelial cell adhesion to the extracellular matrix. dystroglycan bovine aortic endothelial Dulbecco's modified Eagle's medium platelet endothelial cell adhesion molecule-1 bovine serum albumin polymerase chain reaction 4-morpholineethanesulfonic acid Dystroglycan (DG)1consists of αDG and βDG, the two subunits yielded by proteolytic cleavage of a single precursor protein. αDG is a highly glycosylated extracellular protein with a molecular mass of 120–190 kDa, and βDG is a 43-kDa transmembrane protein (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1182) Google Scholar, 2Ibraghimov-Beskrovnaya O. Milatovich A. Ozcelik T. Yang B. Koepnick K. Francke U. Campbell K.P. Hum. Mol. Genet. 1993; 2: 1651-1657Crossref PubMed Scopus (207) Google Scholar). On the cell surface, αDG is anchored to βDG by noncovalent bonds (3Yoshida M. Suzuki A. Yamamoto H. Noguchi S. Mizuno Y. Ozawa E. Eur. J. Biochem. 1994; 222: 1055-1061Crossref PubMed Scopus (190) Google Scholar, 4Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar).DG is the central component of the dystrophin-associated glycoprotein complex, and its physiological roles have been extensively studied in skeletal muscle, from which it was originally isolated. In the skeletal muscle sarcolemma, DG forms a physical link between the extracellular matrix and intracellular cytoskeleton by the binding of αDG and βDG with laminin-2 in the matrix and dystrophin in the cytoskeleton, respectively (5Suzuki A. Yoshida M. Hayashi K. Mizuno Y. Hagiwara Y. Ozawa E. Eur. J. Biochem. 1994; 220: 283-292Crossref PubMed Scopus (219) Google Scholar, 6Henry M.D. Campbell K.P. Curr. Opin. Cell Biol. 1996; 8: 625-631Crossref PubMed Scopus (237) Google Scholar). The membrane stability of the sarcolemma depends on this link as evidenced by the progressive muscle degeneration in Duchnne's muscular dystrophy that is caused by an abnormal dystrophin gene (7Campbell K.P. Cell. 1995; 80: 675-679Abstract Full Text PDF PubMed Scopus (753) Google Scholar). A specialized form of the DG complex is found in the neuromuscular junction, where αDG and βDG associate with agrin in the matrix and utrophin in the cytoskeleton, respectively (8Bowe M.A. Deyst K.A. Leszyk J.D. Fallon J.F. Neuron. 1994; 12: 1173-1180Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 9Gee S.H. Montanaro F. Lindenbaum M.H. Carbonetto S. Cell. 1994; 77: 675-686Abstract Full Text PDF PubMed Scopus (449) Google Scholar, 10Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Abstract Full Text PDF PubMed Scopus (234) Google Scholar). This protein complex is critically involved in agrin-induced clustering of acetylcholine receptors (11Campanelli J.T. Roberds S.L. Campbell K.P. Scheller R.H. Cell. 1994; 77: 663-674Abstract Full Text PDF PubMed Scopus (340) Google Scholar).DG is expressed in various tissues and cell lines, and evidence for its functions in non-muscle tissues has been accumulating in recent years. In the peripheral nervous system, DG is expressed by Schwann cells and is involved both in Schwann cell adhesion to the extracellular matrix and in myelinogenesis (12Yamada H. Shimizu T. Tanaka T. Campbell K.P. Matsumura K. FEBS Lett. 1994; 352: 49-53Crossref PubMed Scopus (148) Google Scholar, 13Yamada H. Chiba A. Endo T. Kobata A. Anderson L.V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Crossref PubMed Scopus (100) Google Scholar, 14Yamada H. Denzer A.J. Hori H. Tanaka T. Anderson L.V.B. Fujita S. Fukuta-Ohi H. Shimizu T. Ruegg M.A. Matsumura K. J. Biol. Chem. 1996; 271: 23418-23423Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In the central nervous system, DG is expressed both by glial cells and by certain groups of neurons and is suggested to be involved in blood brain barrier and synapse formation (16Tian M. Jacobson C. Gee S.H. Campbell K.P. Carbonetto S. Jucker M. Eur. J. Neurosci. 1996; 8: 2739-2747Crossref PubMed Scopus (129) Google Scholar, 17Drenckhahn D. Holbach M. Ness W. Schmitz F. Anderson L.V. Neuroscience. 1996; 73: 605-612Crossref PubMed Scopus (44) Google Scholar, 18Powell S.K. Kleinman H.K. Int. J. Biochem. Cell Biol. 1997; 29: 401-414Crossref PubMed Scopus (159) Google Scholar). Outside the nervous system, DG has been shown to be involved in epithelial morphogenesis during embryogenesis (19Durbeej M. Ekblom P. Exp. Lung Res. 1997; 23: 109-118Crossref PubMed Scopus (41) Google Scholar, 20Durbeej M. Larsson E. Ibraghimov-Beskrovnaya O. Roberds S.L. Campbell K.P. Ekblom P. J. Cell Biol. 1995; 130: 79-91Crossref PubMed Scopus (187) Google Scholar).Immunohistochemical studies using human brain sections indicated the expression of DG by vascular endothelial cells (21Uchino M. Hara A. Mizuno Y. Fujiki M. Nakamura T. Tokunaga M. Hirano T. Yamashita T. Uyama E. Ando Y. Mita S. Ando M. Intern. Med. 1996; 35: 189-194Crossref PubMed Scopus (27) Google Scholar, 22Yamamoto T. Shibata N. Kanazawa M. Kobayashi M. Komori T. Ikeya K. Kondo E. Saito K. Ozawa M. Acta Neuropathol. 1997; 94: 173-179Crossref PubMed Scopus (16) Google Scholar). An immunocytochemical study indicated the expression of DG by cultured human umbilical endothelial cells as well (23Belkin A.M. Smalheiser N.R. Cell Adhes. Commun. 1996; 4: 281-296Crossref PubMed Scopus (43) Google Scholar). The expression of DG by vascular endothelial cells, however, is still controversial because of the negative immunostaining of anti-DG antibodies found in brain capillary endothelial cells (16Tian M. Jacobson C. Gee S.H. Campbell K.P. Carbonetto S. Jucker M. Eur. J. Neurosci. 1996; 8: 2739-2747Crossref PubMed Scopus (129) Google Scholar). Most recently, Durbeej et al. (24Durbeej M. Henry M.D. Ferletta M. Campbell K.P. Ekblom P. J. Histochem. Cytochem. 1998; 46: 449-457Crossref PubMed Scopus (154) Google Scholar) suggested that the anti-DG immunostaining in some blood vessels emanates not from the endothelial cells, but from the smooth muscle cells, which are a rich source of DG.Vascular endothelial cells undergo drastic morphological and functional changes during angiogenesis, and it is well established that the behavior of the cell is critically influenced by interaction with the extracellular matrix in their milieu. Laminin is the major constituent of the vascular endothelial basement membrane (25Sanes J. Engvall E. Butkowski R. Hunter D.D. J. Cell Biol. 1990; 111: 1685-1699Crossref PubMed Scopus (499) Google Scholar), and it is generally accepted that the principal endothelial cell-surface receptor that recognizes laminin is the integrin family of cell adhesion molecules (26Languino L.R. Gehlsen K.R. Wayner E. Carter W.G. Engvall E. Ruoslahti E. J. Cell Biol. 1989; 109: 2455-2462Crossref PubMed Scopus (258) Google Scholar, 27Cheng Y.F. Kramer R.H. J. Cell. Physiol. 1989; 139: 275-286Crossref PubMed Scopus (74) Google Scholar, 28Albelda S.M. Daise M. Levine E.M. Buck C.A. J. Clin. Invest. 1989; 83: 1992-2002Crossref PubMed Scopus (143) Google Scholar). Several lines of evidence, however, suggested the existence of non-integrin types of laminin receptors expressed by vascular endothelial cells whose identity remains unclear (29Tressler R.J. Belloni P.N. Nicolson G.L. Cancer Commun. 1989; 1: 55-63Crossref PubMed Scopus (48) Google Scholar, 30Basson C.T. Knowles W.J. Bell L. Albelda S.M. Castronovo V. Liotta L.A. Madri J.A. J. Cell Biol. 1990; 110: 789-801Crossref PubMed Scopus (74) Google Scholar). Given the laminin-binding capacity of DG, the purposes of this study were to confirm the expression of DG by primary cultured bovine aortic endothelial (BAE) cells and to establish the role of DG as a non-integrin type of laminin receptor involved in BAE cell adhesion to the extracellular matrix.DISCUSSIONIn this study, expression of DG by cultured BAE cells was confirmed by cDNA cloning from a BAE cDNA library, Northern blotting of mRNA, and Western blotting of membrane proteins (Fig.1, a and b). Coexpression of βDG and PECAM1 by the cells confirmed the endothelial cell origin of the DG mRNA and protein detected (Fig. 1 c). The length of the deduced amino acid sequence of bovine DG (895 amino acids) was exactly the same as those of the rabbit, mouse, and human versions, and 91–93% of the amino acid sequence was identical to the three reported sequences (6Henry M.D. Campbell K.P. Curr. Opin. Cell Biol. 1996; 8: 625-631Crossref PubMed Scopus (237) Google Scholar). Together with the similar size of the mRNA and its ubiquitous distribution on the Northern blot, these results indicate high conservation of the DG precursor gene among mammalian species.Immunocytochemical analysis revealed localization of DG on the basal side of BAE cells and a drastic change in the localization associated with cell migration (Fig. 2). In the resting cells, DG was confined in multiple plaques, the morphology of which is apparently close to that of the laminin/agrin-induced DG plaques in skeletal muscle cells (41Cohen M.W. Jacobson C. Yurchenco P.D. Morris G.E. Carbonetto S. J. Cell Biol. 1997; 136: 1047-1058Crossref PubMed Scopus (71) Google Scholar). The plaques were obscured in migrating cells, in which the trailing edge was most intensely stained. The trailing edge was then retracted when the cells actually moved in space. A straightforward explanation for these observations is that DG, with its tight association with the extracellular matrix, is left in the last part of the cell that detaches from the substratum when it moves. Although it is necessary to monitor the location of DG in live cells to verify this scenario, it is at least clear that the subcellular localization of DG in migrating BAE cells is quite different from that of integrins that have been shown to be recruited to the frontal portion of migrating leukocytes (42Lawson M.A. Maxfield F.R. Nature. 1995; 377: 75-79Crossref PubMed Scopus (477) Google Scholar).Two lines of biochemical evidence were presented in the this study that indicated the role of DG as a non-integrin laminin receptor involved in BAE cell adhesion to the extracellular matrix. The first is the inhibition of BAE cell adhesion to laminin-1 by soluble αDG contained in the conditioned medium from DG cDNA-transfected cells (Fig. 6). Soluble αDG could also inhibit the biotin-laminin-1 binding to αDG in the dissociated membrane proteins (Fig. 3). Secretion of αDG to the medium was first indicated in RT4 schwannoma cells (15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and we have confirmed the finding in cultured BAE cells. The molecular mechanism that generates the soluble form and its physiological role must be the subjects in a future study.The second line of biochemical evidence is the inhibition of BAE cell adhesion to laminin-1 by a set of glycosaminoglycans (heparin, dextran sulfate, fucoidan, and sulfatide) (Fig. 7). Of the four glycosaminoglycans, sulfatide was by far the most effective; however, it did not inhibit the αDG-laminin-1 interaction in the ligand overlay assay (Fig. 4), and it also inhibited the BAE cell adhesion to fibronectin (Fig. 7). Therefore, it is plausible to conclude that the other three, but not sulfatide, inhibited BAE cell adhesion to laminin-1 by specifically disrupting the αDG-laminin-1 interaction. The mechanism for sulfatide inhibition is still unknown. The three glycosaminoglycans (heparin, dextran sulfate, and fucoidan) have been shown to inhibit the adhesion of RT4 schwannoma cells to laminin-1 (15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) as well as agrin-induced acetylcholine receptor clustering in myotubes (43Bruce G.W. J. Neurosci. 1990; 10: 3576-3582Crossref PubMed Google Scholar), suggesting that the similar carbohydrate moiety on αDG was involved in the binding to laminin-1/agrin in these cell lines. Together with the failure of oligosaccharides to inhibit the adhesion of RT4 (15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and BAE (Fig. 7) cells, these results indicated the importance of the high anionic charge and polymeric structure of the glycosaminoglycans in interrupting the αDG-laminin-1 interaction. The soluble αDG in the conditioned medium caused no further decrement of the cell adhesion in the presence heparin (Fig. 6), providing supportive evidence for the interruption of the αDG-laminin-1 interaction by heparin.Of the three glycosaminoglycans, heparin has been known for its activities to stimulate both endothelial cell proliferation (44Thornton S.C. Mueller S.N. Levine E.M. Science. 1983; 222: 623-625Crossref PubMed Scopus (582) Google Scholar) and migration (45Azizkhan R.G. Azizkhan J.C. Zetter B.R. Folkman J. J. Exp. Med. 1980; 152: 931-934Crossref PubMed Scopus (337) Google Scholar). Polyanions such as heparin bind to a wide variety of glycoproteins, including extracellular matrix proteins, growth factors, and protease inhibitors, and the mechanism of action of heparin to modulate endothelial cell behavior is still unknown. Studies are underway in our laboratory to test the hypothesis that the cell behavior modulation by heparin is due to the inhibition of the αDG-laminin interaction.Both in the cases of the soluble αDG and the three glycosaminoglycans, inhibition was not observed in fibronectin-coated dishes, suggesting the laminin specificity of the DG-mediated adhesion. Laminin is a group of heterogeneous proteins, and care must be taken to interpret the results obtained with a purified protein. The mouse Engelbreth-Holm-Swarm laminin-1 used in this study is composed of the three subunits, α1, β1, and γ1. BAE cells have been shown to express β1 and γ1, but not α1 (40Sorokin L. Girg W. Gopfert T. Hallmann R. Deutzmann R. Eur. J. Biochem. 1994; 223: 603-610Crossref PubMed Scopus (72) Google Scholar). Recent histochemical analysis indicated the presence of α5, but not α1, in the vascular basal laminae of murine heart (23Belkin A.M. Smalheiser N.R. Cell Adhes. Commun. 1996; 4: 281-296Crossref PubMed Scopus (43) Google Scholar). The laminin α5 in the vascular basal laminae may be produced by the underlying smooth muscle cells because neither BAE nor mouse aortic endothelial cells produce laminin α5, but only laminin α4 (46Frieser M. Nockel H. Pausch F. Roder C. Hahn A. Deutzmann R. Sorokin L.M. Eur. J. Biochem. 1997; 246: 727-735Crossref PubMed Scopus (111) Google Scholar). Therefore, it is at least clear that laminin-1 is unlikely to be an in vivo ligand for DG in vascular endothelial cells. We have, however, shown in the ligand overlay assay that the α5 protein expressed in E. colibound αDG and that it could disrupt the binding of laminin-1 to αDG (Fig. 5). The recombinant α5 protein immobilized on the dishes promoted the BAE cell adhesion (data not shown). These results support the idea that endothelial DG works as a laminin receptor in vivo. Further studies with laminin α5 purified from tissues or gene knockout experiments will be required to test the idea directly.In conclusion, we have confirmed the expression of DG by cultured BAE cells and presented evidence for the role of DG as a non-integrin laminin receptor involved in BAE cell adhesion to the extracellular matrix. These findings expand our knowledge on the physiological roles of DG in non-muscle tissues. The distinct subcellular localization of DG in BAE cells and the essential role of the carbohydrate moiety in the αDG-laminin interaction suggest the influence of DG-mediated cell adhesion that is quite different from that of integrin-mediated cell adhesion. The control of cell behavior by the DG-laminin interaction must be the subject for future studies. Dystroglycan (DG)1consists of αDG and βDG, the two subunits yielded by proteolytic cleavage of a single precursor protein. αDG is a highly glycosylated extracellular protein with a molecular mass of 120–190 kDa, and βDG is a 43-kDa transmembrane protein (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Nature. 1992; 355: 696-702Crossref PubMed Scopus (1182) Google Scholar, 2Ibraghimov-Beskrovnaya O. Milatovich A. Ozcelik T. Yang B. Koepnick K. Francke U. Campbell K.P. Hum. Mol. Genet. 1993; 2: 1651-1657Crossref PubMed Scopus (207) Google Scholar). On the cell surface, αDG is anchored to βDG by noncovalent bonds (3Yoshida M. Suzuki A. Yamamoto H. Noguchi S. Mizuno Y. Ozawa E. Eur. J. Biochem. 1994; 222: 1055-1061Crossref PubMed Scopus (190) Google Scholar, 4Jung D. Yang B. Meyer J. Chamberlain J.S. Campbell K.P. J. Biol. Chem. 1995; 270: 27305-27310Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). DG is the central component of the dystrophin-associated glycoprotein complex, and its physiological roles have been extensively studied in skeletal muscle, from which it was originally isolated. In the skeletal muscle sarcolemma, DG forms a physical link between the extracellular matrix and intracellular cytoskeleton by the binding of αDG and βDG with laminin-2 in the matrix and dystrophin in the cytoskeleton, respectively (5Suzuki A. Yoshida M. Hayashi K. Mizuno Y. Hagiwara Y. Ozawa E. Eur. J. Biochem. 1994; 220: 283-292Crossref PubMed Scopus (219) Google Scholar, 6Henry M.D. Campbell K.P. Curr. Opin. Cell Biol. 1996; 8: 625-631Crossref PubMed Scopus (237) Google Scholar). The membrane stability of the sarcolemma depends on this link as evidenced by the progressive muscle degeneration in Duchnne's muscular dystrophy that is caused by an abnormal dystrophin gene (7Campbell K.P. Cell. 1995; 80: 675-679Abstract Full Text PDF PubMed Scopus (753) Google Scholar). A specialized form of the DG complex is found in the neuromuscular junction, where αDG and βDG associate with agrin in the matrix and utrophin in the cytoskeleton, respectively (8Bowe M.A. Deyst K.A. Leszyk J.D. Fallon J.F. Neuron. 1994; 12: 1173-1180Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 9Gee S.H. Montanaro F. Lindenbaum M.H. Carbonetto S. Cell. 1994; 77: 675-686Abstract Full Text PDF PubMed Scopus (449) Google Scholar, 10Sugiyama J. Bowen D.C. Hall Z.W. Neuron. 1994; 13: 103-115Abstract Full Text PDF PubMed Scopus (234) Google Scholar). This protein complex is critically involved in agrin-induced clustering of acetylcholine receptors (11Campanelli J.T. Roberds S.L. Campbell K.P. Scheller R.H. Cell. 1994; 77: 663-674Abstract Full Text PDF PubMed Scopus (340) Google Scholar). DG is expressed in various tissues and cell lines, and evidence for its functions in non-muscle tissues has been accumulating in recent years. In the peripheral nervous system, DG is expressed by Schwann cells and is involved both in Schwann cell adhesion to the extracellular matrix and in myelinogenesis (12Yamada H. Shimizu T. Tanaka T. Campbell K.P. Matsumura K. FEBS Lett. 1994; 352: 49-53Crossref PubMed Scopus (148) Google Scholar, 13Yamada H. Chiba A. Endo T. Kobata A. Anderson L.V.B. Hori H. Fukuta-Ohi H. Kanazawa I. Campbell K.P. Shimizu T. Matsumura K. J. Neurochem. 1996; 66: 1518-1524Crossref PubMed Scopus (100) Google Scholar, 14Yamada H. Denzer A.J. Hori H. Tanaka T. Anderson L.V.B. Fujita S. Fukuta-Ohi H. Shimizu T. Ruegg M.A. Matsumura K. J. Biol. Chem. 1996; 271: 23418-23423Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In the central nervous system, DG is expressed both by glial cells and by certain groups of neurons and is suggested to be involved in blood brain barrier and synapse formation (16Tian M. Jacobson C. Gee S.H. Campbell K.P. Carbonetto S. Jucker M. Eur. J. Neurosci. 1996; 8: 2739-2747Crossref PubMed Scopus (129) Google Scholar, 17Drenckhahn D. Holbach M. Ness W. Schmitz F. Anderson L.V. Neuroscience. 1996; 73: 605-612Crossref PubMed Scopus (44) Google Scholar, 18Powell S.K. Kleinman H.K. Int. J. Biochem. Cell Biol. 1997; 29: 401-414Crossref PubMed Scopus (159) Google Scholar). Outside the nervous system, DG has been shown to be involved in epithelial morphogenesis during embryogenesis (19Durbeej M. Ekblom P. Exp. Lung Res. 1997; 23: 109-118Crossref PubMed Scopus (41) Google Scholar, 20Durbeej M. Larsson E. Ibraghimov-Beskrovnaya O. Roberds S.L. Campbell K.P. Ekblom P. J. Cell Biol. 1995; 130: 79-91Crossref PubMed Scopus (187) Google Scholar). Immunohistochemical studies using human brain sections indicated the expression of DG by vascular endothelial cells (21Uchino M. Hara A. Mizuno Y. Fujiki M. Nakamura T. Tokunaga M. Hirano T. Yamashita T. Uyama E. Ando Y. Mita S. Ando M. Intern. Med. 1996; 35: 189-194Crossref PubMed Scopus (27) Google Scholar, 22Yamamoto T. Shibata N. Kanazawa M. Kobayashi M. Komori T. Ikeya K. Kondo E. Saito K. Ozawa M. Acta Neuropathol. 1997; 94: 173-179Crossref PubMed Scopus (16) Google Scholar). An immunocytochemical study indicated the expression of DG by cultured human umbilical endothelial cells as well (23Belkin A.M. Smalheiser N.R. Cell Adhes. Commun. 1996; 4: 281-296Crossref PubMed Scopus (43) Google Scholar). The expression of DG by vascular endothelial cells, however, is still controversial because of the negative immunostaining of anti-DG antibodies found in brain capillary endothelial cells (16Tian M. Jacobson C. Gee S.H. Campbell K.P. Carbonetto S. Jucker M. Eur. J. Neurosci. 1996; 8: 2739-2747Crossref PubMed Scopus (129) Google Scholar). Most recently, Durbeej et al. (24Durbeej M. Henry M.D. Ferletta M. Campbell K.P. Ekblom P. J. Histochem. Cytochem. 1998; 46: 449-457Crossref PubMed Scopus (154) Google Scholar) suggested that the anti-DG immunostaining in some blood vessels emanates not from the endothelial cells, but from the smooth muscle cells, which are a rich source of DG. Vascular endothelial cells undergo drastic morphological and functional changes during angiogenesis, and it is well established that the behavior of the cell is critically influenced by interaction with the extracellular matrix in their milieu. Laminin is the major constituent of the vascular endothelial basement membrane (25Sanes J. Engvall E. Butkowski R. Hunter D.D. J. Cell Biol. 1990; 111: 1685-1699Crossref PubMed Scopus (499) Google Scholar), and it is generally accepted that the principal endothelial cell-surface receptor that recognizes laminin is the integrin family of cell adhesion molecules (26Languino L.R. Gehlsen K.R. Wayner E. Carter W.G. Engvall E. Ruoslahti E. J. Cell Biol. 1989; 109: 2455-2462Crossref PubMed Scopus (258) Google Scholar, 27Cheng Y.F. Kramer R.H. J. Cell. Physiol. 1989; 139: 275-286Crossref PubMed Scopus (74) Google Scholar, 28Albelda S.M. Daise M. Levine E.M. Buck C.A. J. Clin. Invest. 1989; 83: 1992-2002Crossref PubMed Scopus (143) Google Scholar). Several lines of evidence, however, suggested the existence of non-integrin types of laminin receptors expressed by vascular endothelial cells whose identity remains unclear (29Tressler R.J. Belloni P.N. Nicolson G.L. Cancer Commun. 1989; 1: 55-63Crossref PubMed Scopus (48) Google Scholar, 30Basson C.T. Knowles W.J. Bell L. Albelda S.M. Castronovo V. Liotta L.A. Madri J.A. J. Cell Biol. 1990; 110: 789-801Crossref PubMed Scopus (74) Google Scholar). Given the laminin-binding capacity of DG, the purposes of this study were to confirm the expression of DG by primary cultured bovine aortic endothelial (BAE) cells and to establish the role of DG as a non-integrin type of laminin receptor involved in BAE cell adhesion to the extracellular matrix. DISCUSSIONIn this study, expression of DG by cultured BAE cells was confirmed by cDNA cloning from a BAE cDNA library, Northern blotting of mRNA, and Western blotting of membrane proteins (Fig.1, a and b). Coexpression of βDG and PECAM1 by the cells confirmed the endothelial cell origin of the DG mRNA and protein detected (Fig. 1 c). The length of the deduced amino acid sequence of bovine DG (895 amino acids) was exactly the same as those of the rabbit, mouse, and human versions, and 91–93% of the amino acid sequence was identical to the three reported sequences (6Henry M.D. Campbell K.P. Curr. Opin. Cell Biol. 1996; 8: 625-631Crossref PubMed Scopus (237) Google Scholar). Together with the similar size of the mRNA and its ubiquitous distribution on the Northern blot, these results indicate high conservation of the DG precursor gene among mammalian species.Immunocytochemical analysis revealed localization of DG on the basal side of BAE cells and a drastic change in the localization associated with cell migration (Fig. 2). In the resting cells, DG was confined in multiple plaques, the morphology of which is apparently close to that of the laminin/agrin-induced DG plaques in skeletal muscle cells (41Cohen M.W. Jacobson C. Yurchenco P.D. Morris G.E. Carbonetto S. J. Cell Biol. 1997; 136: 1047-1058Crossref PubMed Scopus (71) Google Scholar). The plaques were obscured in migrating cells, in which the trailing edge was most intensely stained. The trailing edge was then retracted when the cells actually moved in space. A straightforward explanation for these observations is that DG, with its tight association with the extracellular matrix, is left in the last part of the cell that detaches from the substratum when it moves. Although it is necessary to monitor the location of DG in live cells to verify this scenario, it is at least clear that the subcellular localization of DG in migrating BAE cells is quite different from that of integrins that have been shown to be recruited to the frontal portion of migrating leukocytes (42Lawson M.A. Maxfield F.R. Nature. 1995; 377: 75-79Crossref PubMed Scopus (477) Google Scholar).Two lines of biochemical evidence were presented in the this study that indicated the role of DG as a non-integrin laminin receptor involved in BAE cell adhesion to the extracellular matrix. The first is the inhibition of BAE cell adhesion to laminin-1 by soluble αDG contained in the conditioned medium from DG cDNA-transfected cells (Fig. 6). Soluble αDG could also inhibit the biotin-laminin-1 binding to αDG in the dissociated membrane proteins (Fig. 3). Secretion of αDG to the medium was first indicated in RT4 schwannoma cells (15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and we have confirmed the finding in cultured BAE cells. The molecular mechanism that generates the soluble form and its physiological role must be the subjects in a future study.The second line of biochemical evidence is the inhibition of BAE cell adhesion to laminin-1 by a set of glycosaminoglycans (heparin, dextran sulfate, fucoidan, and sulfatide) (Fig. 7). Of the four glycosaminoglycans, sulfatide was by far the most effective; however, it did not inhibit the αDG-laminin-1 interaction in the ligand overlay assay (Fig. 4), and it also inhibited the BAE cell adhesion to fibronectin (Fig. 7). Therefore, it is plausible to conclude that the other three, but not sulfatide, inhibited BAE cell adhesion to laminin-1 by specifically disrupting the αDG-laminin-1 interaction. The mechanism for sulfatide inhibition is still unknown. The three glycosaminoglycans (heparin, dextran sulfate, and fucoidan) have been shown to inhibit the adhesion of RT4 schwannoma cells to laminin-1 (15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) as well as agrin-induced acetylcholine receptor clustering in myotubes (43Bruce G.W. J. Neurosci. 1990; 10: 3576-3582Crossref PubMed Google Scholar), suggesting that the similar carbohydrate moiety on αDG was involved in the binding to laminin-1/agrin in these cell lines. Together with the failure of oligosaccharides to inhibit the adhesion of RT4 (15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and BAE (Fig. 7) cells, these results indicated the importance of the high anionic charge and polymeric structure of the glycosaminoglycans in interrupting the αDG-laminin-1 interaction. The soluble αDG in the conditioned medium caused no further decrement of the cell adhesion in the presence heparin (Fig. 6), providing supportive evidence for the interruption of the αDG-laminin-1 interaction by heparin.Of the three glycosaminoglycans, heparin has been known for its activities to stimulate both endothelial cell proliferation (44Thornton S.C. Mueller S.N. Levine E.M. Science. 1983; 222: 623-625Crossref PubMed Scopus (582) Google Scholar) and migration (45Azizkhan R.G. Azizkhan J.C. Zetter B.R. Folkman J. J. Exp. Med. 1980; 152: 931-934Crossref PubMed Scopus (337) Google Scholar). Polyanions such as heparin bind to a wide variety of glycoproteins, including extracellular matrix proteins, growth factors, and protease inhibitors, and the mechanism of action of heparin to modulate endothelial cell behavior is still unknown. Studies are underway in our laboratory to test the hypothesis that the cell behavior modulation by heparin is due to the inhibition of the αDG-laminin interaction.Both in the cases of the soluble αDG and the three glycosaminoglycans, inhibition was not observed in fibronectin-coated dishes, suggesting the laminin specificity of the DG-mediated adhesion. Laminin is a group of heterogeneous proteins, and care must be taken to interpret the results obtained with a purified protein. The mouse Engelbreth-Holm-Swarm laminin-1 used in this study is composed of the three subunits, α1, β1, and γ1. BAE cells have been shown to express β1 and γ1, but not α1 (40Sorokin L. Girg W. Gopfert T. Hallmann R. Deutzmann R. Eur. J. Biochem. 1994; 223: 603-610Crossref PubMed Scopus (72) Google Scholar). Recent histochemical analysis indicated the presence of α5, but not α1, in the vascular basal laminae of murine heart (23Belkin A.M. Smalheiser N.R. Cell Adhes. Commun. 1996; 4: 281-296Crossref PubMed Scopus (43) Google Scholar). The laminin α5 in the vascular basal laminae may be produced by the underlying smooth muscle cells because neither BAE nor mouse aortic endothelial cells produce laminin α5, but only laminin α4 (46Frieser M. Nockel H. Pausch F. Roder C. Hahn A. Deutzmann R. Sorokin L.M. Eur. J. Biochem. 1997; 246: 727-735Crossref PubMed Scopus (111) Google Scholar). Therefore, it is at least clear that laminin-1 is unlikely to be an in vivo ligand for DG in vascular endothelial cells. We have, however, shown in the ligand overlay assay that the α5 protein expressed in E. colibound αDG and that it could disrupt the binding of laminin-1 to αDG (Fig. 5). The recombinant α5 protein immobilized on the dishes promoted the BAE cell adhesion (data not shown). These results support the idea that endothelial DG works as a laminin receptor in vivo. Further studies with laminin α5 purified from tissues or gene knockout experiments will be required to test the idea directly.In conclusion, we have confirmed the expression of DG by cultured BAE cells and presented evidence for the role of DG as a non-integrin laminin receptor involved in BAE cell adhesion to the extracellular matrix. These findings expand our knowledge on the physiological roles of DG in non-muscle tissues. The distinct subcellular localization of DG in BAE cells and the essential role of the carbohydrate moiety in the αDG-laminin interaction suggest the influence of DG-mediated cell adhesion that is quite different from that of integrin-mediated cell adhesion. The control of cell behavior by the DG-laminin interaction must be the subject for future studies. In this study, expression of DG by cultured BAE cells was confirmed by cDNA cloning from a BAE cDNA library, Northern blotting of mRNA, and Western blotting of membrane proteins (Fig.1, a and b). Coexpression of βDG and PECAM1 by the cells confirmed the endothelial cell origin of the DG mRNA and protein detected (Fig. 1 c). The length of the deduced amino acid sequence of bovine DG (895 amino acids) was exactly the same as those of the rabbit, mouse, and human versions, and 91–93% of the amino acid sequence was identical to the three reported sequences (6Henry M.D. Campbell K.P. Curr. Opin. Cell Biol. 1996; 8: 625-631Crossref PubMed Scopus (237) Google Scholar). Together with the similar size of the mRNA and its ubiquitous distribution on the Northern blot, these results indicate high conservation of the DG precursor gene among mammalian species. Immunocytochemical analysis revealed localization of DG on the basal side of BAE cells and a drastic change in the localization associated with cell migration (Fig. 2). In the resting cells, DG was confined in multiple plaques, the morphology of which is apparently close to that of the laminin/agrin-induced DG plaques in skeletal muscle cells (41Cohen M.W. Jacobson C. Yurchenco P.D. Morris G.E. Carbonetto S. J. Cell Biol. 1997; 136: 1047-1058Crossref PubMed Scopus (71) Google Scholar). The plaques were obscured in migrating cells, in which the trailing edge was most intensely stained. The trailing edge was then retracted when the cells actually moved in space. A straightforward explanation for these observations is that DG, with its tight association with the extracellular matrix, is left in the last part of the cell that detaches from the substratum when it moves. Although it is necessary to monitor the location of DG in live cells to verify this scenario, it is at least clear that the subcellular localization of DG in migrating BAE cells is quite different from that of integrins that have been shown to be recruited to the frontal portion of migrating leukocytes (42Lawson M.A. Maxfield F.R. Nature. 1995; 377: 75-79Crossref PubMed Scopus (477) Google Scholar). Two lines of biochemical evidence were presented in the this study that indicated the role of DG as a non-integrin laminin receptor involved in BAE cell adhesion to the extracellular matrix. The first is the inhibition of BAE cell adhesion to laminin-1 by soluble αDG contained in the conditioned medium from DG cDNA-transfected cells (Fig. 6). Soluble αDG could also inhibit the biotin-laminin-1 binding to αDG in the dissociated membrane proteins (Fig. 3). Secretion of αDG to the medium was first indicated in RT4 schwannoma cells (15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and we have confirmed the finding in cultured BAE cells. The molecular mechanism that generates the soluble form and its physiological role must be the subjects in a future study. The second line of biochemical evidence is the inhibition of BAE cell adhesion to laminin-1 by a set of glycosaminoglycans (heparin, dextran sulfate, fucoidan, and sulfatide) (Fig. 7). Of the four glycosaminoglycans, sulfatide was by far the most effective; however, it did not inhibit the αDG-laminin-1 interaction in the ligand overlay assay (Fig. 4), and it also inhibited the BAE cell adhesion to fibronectin (Fig. 7). Therefore, it is plausible to conclude that the other three, but not sulfatide, inhibited BAE cell adhesion to laminin-1 by specifically disrupting the αDG-laminin-1 interaction. The mechanism for sulfatide inhibition is still unknown. The three glycosaminoglycans (heparin, dextran sulfate, and fucoidan) have been shown to inhibit the adhesion of RT4 schwannoma cells to laminin-1 (15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) as well as agrin-induced acetylcholine receptor clustering in myotubes (43Bruce G.W. J. Neurosci. 1990; 10: 3576-3582Crossref PubMed Google Scholar), suggesting that the similar carbohydrate moiety on αDG was involved in the binding to laminin-1/agrin in these cell lines. Together with the failure of oligosaccharides to inhibit the adhesion of RT4 (15Matsumura K. Chiba A. Yamada H. Fukuta-Ohi H. Fujita S. Endo T. Kobata A. Anderson L.V.B. Kanazawa I. Campbell K.P. Shimizu T. J. Biol. Chem. 1997; 272: 13904-13910Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and BAE (Fig. 7) cells, these results indicated the importance of the high anionic charge and polymeric structure of the glycosaminoglycans in interrupting the αDG-laminin-1 interaction. The soluble αDG in the conditioned medium caused no further decrement of the cell adhesion in the presence heparin (Fig. 6), providing supportive evidence for the interruption of the αDG-laminin-1 interaction by heparin. Of the three glycosaminoglycans, heparin has been known for its activities to stimulate both endothelial cell proliferation (44Thornton S.C. Mueller S.N. Levine E.M. Science. 1983; 222: 623-625Crossref PubMed Scopus (582) Google Scholar) and migration (45Azizkhan R.G. Azizkhan J.C. Zetter B.R. Folkman J. J. Exp. Med. 1980; 152: 931-934Crossref PubMed Scopus (337) Google Scholar). Polyanions such as heparin bind to a wide variety of glycoproteins, including extracellular matrix proteins, growth factors, and protease inhibitors, and the mechanism of action of heparin to modulate endothelial cell behavior is still unknown. Studies are underway in our laboratory to test the hypothesis that the cell behavior modulation by heparin is due to the inhibition of the αDG-laminin interaction. Both in the cases of the soluble αDG and the three glycosaminoglycans, inhibition was not observed in fibronectin-coated dishes, suggesting the laminin specificity of the DG-mediated adhesion. Laminin is a group of heterogeneous proteins, and care must be taken to interpret the results obtained with a purified protein. The mouse Engelbreth-Holm-Swarm laminin-1 used in this study is composed of the three subunits, α1, β1, and γ1. BAE cells have been shown to express β1 and γ1, but not α1 (40Sorokin L. Girg W. Gopfert T. Hallmann R. Deutzmann R. Eur. J. Biochem. 1994; 223: 603-610Crossref PubMed Scopus (72) Google Scholar). Recent histochemical analysis indicated the presence of α5, but not α1, in the vascular basal laminae of murine heart (23Belkin A.M. Smalheiser N.R. Cell Adhes. Commun. 1996; 4: 281-296Crossref PubMed Scopus (43) Google Scholar). The laminin α5 in the vascular basal laminae may be produced by the underlying smooth muscle cells because neither BAE nor mouse aortic endothelial cells produce laminin α5, but only laminin α4 (46Frieser M. Nockel H. Pausch F. Roder C. Hahn A. Deutzmann R. Sorokin L.M. Eur. J. Biochem. 1997; 246: 727-735Crossref PubMed Scopus (111) Google Scholar). Therefore, it is at least clear that laminin-1 is unlikely to be an in vivo ligand for DG in vascular endothelial cells. We have, however, shown in the ligand overlay assay that the α5 protein expressed in E. colibound αDG and that it could disrupt the binding of laminin-1 to αDG (Fig. 5). The recombinant α5 protein immobilized on the dishes promoted the BAE cell adhesion (data not shown). These results support the idea that endothelial DG works as a laminin receptor in vivo. Further studies with laminin α5 purified from tissues or gene knockout experiments will be required to test the idea directly. In conclusion, we have confirmed the expression of DG by cultured BAE cells and presented evidence for the role of DG as a non-integrin laminin receptor involved in BAE cell adhesion to the extracellular matrix. These findings expand our knowledge on the physiological roles of DG in non-muscle tissues. The distinct subcellular localization of DG in BAE cells and the essential role of the carbohydrate moiety in the αDG-laminin interaction suggest the influence of DG-mediated cell adhesion that is quite different from that of integrin-mediated cell adhesion. The control of cell behavior by the DG-laminin interaction must be the subject for future studies." @default.
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W2061004014 title "Adhesion of Cultured Bovine Aortic Endothelial Cells to Laminin-1 Mediated by Dystroglycan" @default.
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