Mapping the Type I Collagen-binding Site on Pigment Epithelium-derived Factor

Abstract

Pigment epithelium-derived factor (PEDF), a neurotrophic and antiangiogenic serpin, is identified in tissues rich in collagen, e.g. cornea, vitreous, bone, and cartilage. We show that recombinant human PEDF formed complexes with collagens from the bovine cornea and vitreous. We have examined the direct binding of PEDF to collagen I and found that interactions were ionic in nature and occurred when PEDF and collagen I were both in solution, when either one was immobilized, or even when collagen I was denatured under reducing conditions. 125I-PEDF bound to immobilized collagen I in a saturable fashion (K D = 123 nm). Compared with neurotrophic PEDF-derived peptides, ovalbumin and angiogenic inhibitors, only full-length PEDF competed efficiently with 125I-PEDF for the binding to immobilized collagen I (EC50 = 3 μg/ml). The collagen-binding region was analyzed using controlled proteolysis and chemically modified PEDF. Cleavage of the serpin exposed loop did not prevent binding to collagen I. Conjugation of lysines with fluorescein increased the collagen binding affinity. However, treatment of PEDF with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide abolished it, implicating the PEDF aspartic and/or glutamic acid residues in its interaction with collagen I. A negatively charged region on the surface of the PEDF molecule is rich in acidic residues (Glu41, Glu42, Glu43, Asp44, Asp64, Asp256, Asp258, Glu290, Glu291, Glu296, Asp300, Glu304) available to interact directly with positively charged areas of collagen. This represents the first collagen-binding site described for a serpin, which in PEDF, is distinct from its heparin-binding region, neurotrophic active site, and its serpin exposed loop. The collagen-binding property of PEDF may play a role in surface localization and modulation of its antiangiogenic effects in the eye and bone. Pigment epithelium-derived factor (PEDF), a neurotrophic and antiangiogenic serpin, is identified in tissues rich in collagen, e.g. cornea, vitreous, bone, and cartilage. We show that recombinant human PEDF formed complexes with collagens from the bovine cornea and vitreous. We have examined the direct binding of PEDF to collagen I and found that interactions were ionic in nature and occurred when PEDF and collagen I were both in solution, when either one was immobilized, or even when collagen I was denatured under reducing conditions. 125I-PEDF bound to immobilized collagen I in a saturable fashion (K D = 123 nm). Compared with neurotrophic PEDF-derived peptides, ovalbumin and angiogenic inhibitors, only full-length PEDF competed efficiently with 125I-PEDF for the binding to immobilized collagen I (EC50 = 3 μg/ml). The collagen-binding region was analyzed using controlled proteolysis and chemically modified PEDF. Cleavage of the serpin exposed loop did not prevent binding to collagen I. Conjugation of lysines with fluorescein increased the collagen binding affinity. However, treatment of PEDF with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide abolished it, implicating the PEDF aspartic and/or glutamic acid residues in its interaction with collagen I. A negatively charged region on the surface of the PEDF molecule is rich in acidic residues (Glu41, Glu42, Glu43, Asp44, Asp64, Asp256, Asp258, Glu290, Glu291, Glu296, Asp300, Glu304) available to interact directly with positively charged areas of collagen. This represents the first collagen-binding site described for a serpin, which in PEDF, is distinct from its heparin-binding region, neurotrophic active site, and its serpin exposed loop. The collagen-binding property of PEDF may play a role in surface localization and modulation of its antiangiogenic effects in the eye and bone. Pigment epithelium-derived factor (PEDF) 1The abbreviations used are: PEDF, pigment epithelium-derived factor; Fl, fluorescein; PBS, phosphate-buffered saline; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbomiide HCl; MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin; NHS, N-hydroxysuccinimide; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is an extracellular protein that has neurotrophic and antiangiogenic activities expressed mainly in compartments of the eye. It acts in neuronal survival and differentiation on photoreceptor cells and on neuronal cells of the retina and central nervous system (1Becerra S.P. Adv. Exp. Med. Biol. 1997; 425: 223-237Google Scholar, 2Cayouette M. Smith S.B. Becerra S.P. Gravel C. Neurobiol. Dis. 1999; 6: 523-532Google Scholar, 3Cao W. Tombran-Tink J. Elias R. Sezate S. Mrazek D. McGinnis J.F. Investig. Ophthalmol. Vis. Sci. 2001; 42: 1646-1652Google Scholar, 4Houenou L.J. D'Costa A.P. Li L. Turgeon V.L. Enyadike C. Alberdi E. Becerra S.P. J. Comp. Neurol. 1999; 412: 506-514Google Scholar, 5Crawford S.E. Stellmach V. Ranalli M. Huang X. Huang L. Volpert O. De Vries G.H. Abramson L.P. Bouck N. J. Cell Sci. 2001; 114: 4421-4428Google Scholar). Several reports show that PEDF is a major inhibitor of neovascularization and is responsible for excluding vessels from invading the cornea, vitreous, and retina (6Bouck N. Trends Mol. Med. 2002; 8: 330-334Google Scholar, 7Dawson D.W. Volpert O.V. Gillis P. Crawford S.E. Xu H. Benedict W. Bouck N.P. Science. 1999; 285: 245-248Google Scholar, 8Stellmach V. Crawford S.E. Zhou W. Bouck N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2593-2597Google Scholar). These biological activities are of great importance for the development, morphology, and vision process in the eye. PEDF is highly secreted by a variety of cells and associates intimately with extracellular matrix (5Crawford S.E. Stellmach V. Ranalli M. Huang X. Huang L. Volpert O. De Vries G.H. Abramson L.P. Bouck N. J. Cell Sci. 2001; 114: 4421-4428Google Scholar, 9Ortego J. Escribano J. Becerra S.P. Coca-Prados M. Investig. Ophthalmol. Vis. Sci. 1996; 37: 2759-2767Google Scholar, 10Kozaki K. Miyaishi O. Koiwai O. Yasui Y. Kashiwai A. Nishikawa Y. Shimizu S. Saga S. J. Biol. Chem. 1998; 273: 15125-15130Google Scholar, 11Perez-Mediavilla L.A. Chew C. Campochiaro P.A. Nickells R.W. Notario V. Zack D.J. Becerra S.P. Biochim. Biophys. Acta. 1998; 1398: 203-214Google Scholar, 12Stratikos E. Alberdi E. Gettins P.G. Becerra S.P. Protein Sci. 1996; 5: 2575-2582Google Scholar). In particular, cells of the retinal pigment epithelium secrete PEDF protein, which associates with the interphotoreceptor matrix consistent with its affinity for binding glycosaminoglycans, e.g. heparin and heparan sulfate (13Wu Y.Q. Notario V. Chader G.J. Becerra S.P. Protein Expression Purif. 1995; 6: 447-456Google Scholar,14Alberdi E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar). Other areas in the eye that contain PEDF include the vitreous gel and the cornea. In the bovine vitreous, PEDF accumulates at 20 nm accounting for <1% of the total protein of cell-free extracts (15Wu Y.Q. Becerra S.P. Investig. Ophthalmol. Vis. Sci. 1996; 37: 1984-1993Google Scholar). In the human cornea, PEDF immunolabeling has been detected in the epithelium and endothelium (16Karakousis P.C. John S.K. Behling K.C. Surace E.M. Smith J.E. Hendrickson A. Tang W.X. Bennett J. Milam A.H. Mol. Vis. 2001; 7: 154-163Google Scholar). Outside of the eye, PEDF has been detected in teeth, bone, and cartilage matrix (10Kozaki K. Miyaishi O. Koiwai O. Yasui Y. Kashiwai A. Nishikawa Y. Shimizu S. Saga S. J. Biol. Chem. 1998; 273: 15125-15130Google Scholar). It is worth noting that in addition to containing PEDF and being rich in collagen, the adult vitreous, cornea, and bone and cartilage matrixes are vessel-free. Collagens comprise a family of 19 proteins with a broad range of structural and physiological functions, which are strictly located in the extracellular space. They are composed of three chains that fold to form at least one triple helical domain. All collagens are present in tissues as homotrimeric and/or heterotrimeric assemblies and have specific tissue distributions and functions, e.g. they are involved in hemostasis, wound healing, cell adhesion, and migration. In the cornea and vitreous they are known to give transparency, strength, and elasticity. Collagen type I is the major protein of the cornea (94% of the total collagen in the bovine cornea) and coassembles in heterotypic fibrils with collagen type V. Collagen type II is the main protein of the vitreous (70–80%) followed by collagens type V/XI and IX (17Bishop P.N. Prog. Retin. Eye Res. 2000; 19: 323-344Google Scholar). Studies on structure-function relationships have revealed that PEDF is a glycoprotein of that of the of (13Wu Y.Q. Notario V. Chader G.J. Becerra S.P. Protein Expression Purif. 1995; 6: 447-456Google Scholar, Chader G.J. Tombran-Tink J. Proc. Natl. Acad. Sci. U. S. A. Scholar, S.P. A. P. Notario V. J. Biol. Chem. 1995; its neurotrophic and antiangiogenic activities are by of its (1Becerra S.P. Adv. Exp. Med. Biol. 1997; 425: 223-237Google Scholar, 4Houenou L.J. D'Costa A.P. Li L. Turgeon V.L. Enyadike C. Alberdi E. Becerra S.P. J. Comp. Neurol. 1999; 412: 506-514Google Scholar, 7Dawson D.W. Volpert O.V. Gillis P. Crawford S.E. Xu H. Benedict W. Bouck N.P. Science. 1999; 285: 245-248Google Scholar, S.P. A. P. Notario V. J. Biol. Chem. 1995; Scholar). The of are for its neurotrophic activities for its antiangiogenic interactions with on the surface of PEDF the for neurotrophic activities E. Becerra S.P. J. Biol. Chem. 1999; Scholar, Alberdi E.M. A. Becerra S.P. Investig. Ophthalmol. Vis. Sci. 2001; 42: Scholar, D. J. Biol. Chem. 2001; Scholar). The binding for are distinct and from for glycosaminoglycans, with for PEDF (K D the D for and in the PEDF protein molecule E. Alberdi E. Gettins P.G. Becerra S.P. Protein Sci. 1996; 5: 2575-2582Google Scholar, E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar, E. Becerra S.P. J. Biol. Chem. 1999; Scholar, Alberdi E.M. A. Becerra S.P. Investig. Ophthalmol. Vis. Sci. 2001; 42: Scholar). The interact with a neurotrophic region acid in the human PEDF heparin to a region rich in and located to the of binding are distinct and from the serpin loop. the antiangiogenic activities of PEDF and with collagen in extracellular was of to the binding proteins for structure-function We have examined the interactions of PEDF with collagens present in bovine and for recombinant human PEDF PEDF peptides, and three chemically modified PEDF have analyzed the binding site of PEDF to collagen I. We may in the antiangiogenic of PEDF. PEDF was from the of cells containing an for human PEDF as described E. Alberdi E. Gettins P.G. Becerra S.P. Protein Sci. 1996; 5: 2575-2582Google Scholar). 125I-PEDF and were as described E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar, Alberdi E.M. A. Becerra S.P. Investig. Ophthalmol. Vis. Sci. 2001; 42: Scholar). Collagen type I from collagen type from human and with collagen I were from Collagen type II from to bovine collagen type I collagen type II and human collagen type and heparin immobilized on were from The to PEDF, was from to fluorescein was from inhibitor and were from for was from and were from was from phosphate-buffered from and were from was from and was from J. were from J. W. of the eye, the were in a of 20 containing at cornea, and with a at for 20 The was from tissue and by at for at and to at for at The was at and extracts were as described (15Wu Y.Q. Becerra S.P. Investig. Ophthalmol. Vis. Sci. 1996; 37: 1984-1993Google Scholar). of the of the eye, the vitreous gel was to a with a and to at for at The was at and These were on the of complexes formed PEDF and proteins by in which PEDF complexes are by a of PEDF of are E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar). PEDF proteins were with either extracts or collagen in PBS, containing and at for of collagen II and was to the of the PEDF complexes were from PEDF with at for followed by three with The was by and by or as described the the of PEDF was using The binding were analyzed using PEDF affinity was as described E. Becerra S.P. J. Biol. Chem. 1999; Scholar). A total of of PEDF affinity and protein from of collagen and extracts was in an with at for and was by at for at The were three with of was to the at and the was by Protein were by and to The were with in 20 for at with and with for The were with for at with with nm in for at with The were and for with with was detected by with as described for with were with 125I-PEDF in to collagen I immobilized on of at for with the binding was and the were three with was to the at for and to to the of using a binding was from with of PEDF were analyzed by using for I interactions were either of collagen I or PEDF on a by followed by of the proteins to the surface using In the surface of the with to in for The the were on the of the are The protein to bound to the is exposed to the with the on the protein and to the by of the The surface was with and the matrix with and with binding of PEDF or collagen I were in binding with from to and from to and the was to the interaction of both PEDF on a collagen I matrix and the was followed by a The were to binding for a The were with a for the collagen surface binding and with a for the PEDF was with or and of in MES, The were at for the of were to the and to = This was The protein in the PEDF was to heparin affinity E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar). PEDF was in a total of 20 and a of heparin immobilized on The was and the with of S. The bound protein was with a of in The and were using proteolysis of PEDF and was with at a of as described S.P. A. P. Notario V. J. Biol. Chem. 1995; Scholar). Cleavage of the serpin exposed loop was by was using with the followed by either or in were to of 20 of PEDF and fluorescein was by using the the were in a of or of in in or and in and exposed to the were in a of I or in and followed by was with PEDF is a of cornea and vitreous, its to interact with proteins from were at to interactions to with a on a PEDF of was by the of only when with the extracts The of bound PEDF increased with of and protein in the used affinity to proteins in the cornea and vitreous. Several proteins of the cornea and vitreous binding affinity for immobilized PEDF, proteins that with collagen I and with to collagens that the collagen I bound to the PEDF affinity of collagen I that PEDF bound to immobilized denatured collagen I and proteins from vitreous and cornea under reducing These that PEDF to bovine and collagens in and when the binding of PEDF to collagen I and II in that PEDF bound to type of collagen, the serpin The binding was collagen with an for collagen type I the of PEDF binding with of collagen I and II a for a with binding collagen = and collagen = a for the The binding PEDF and collagens occurred at physiological of and and at a with and the of the from to an on the binding with binding at A was for collagen binding not PEDF formed specific complexes with collagens in solution, binding efficiently to type I. PEDF affinity for collagen type I with the analyzed interactions in PEDF with was used as a for collagen I that of the was by in with collagen I in to collagen which with an of PEDF the not as with PEDF, the 125I-PEDF modified on by formed specific complexes with collagen I in the binding of the I binding were with and immobilized collagen I on The binding were under the as the one was that they with of 125I-PEDF at nm bound to the immobilized collagen I and the competed efficiently for the binding with a of = nm A binding was 3 and the binding revealed an affinity of D = nm for the I were with PEDF is a recombinant human PEDF acid S.P. I. A. J. Notario V. Chader G.J. J. Biol. Chem. and the and are from the human PEDF and The the region of PEDF and has neurotrophic on cells E. Becerra S.P. J. Biol. Chem. 1999; Scholar). 3 that of an of the 125I-PEDF with of PEDF. The of in the binding the 125I-PEDF binding to to of the the and did not have a major on the binding of PEDF to collagen I. of or antiangiogenic inhibitors, did not with 125I-PEDF for binding to collagen I. P. collagen I in was used to for of from the immobilized collagen of of collagen I in a in with collagen I 3 and affinity were on the interaction PEDF and collagen I and or binding was with was and for the was in an the PEDF protein was in a interaction the from both and D from with in the was and the collagen was in as the the to effects of one site on a collagen This was consistent with the binding found with collagen in the a range for the for collagen binding to immobilized PEDF, and were consistent with in the collagen I the from the collagen I and the are to of to affinity and of the I not a = = a = = not in a the type of interactions PEDF and collagen examined the of the ionic in the using that the binding of both PEDF and 125I-PEDF to collagen I was with an in The affinity of both PEDF for collagen I with and and was with These that the binding of PEDF to collagen was to ionic strength, an ionic nature for and that the on by with did not binding of PEDF. the ionic nature of the I the role that of the protein play on collagen I The of PEDF has revealed distinct areas with ionic on its surface E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar, M. Gettins P.G. K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: Scholar). In that at the positively charged residues of PEDF abolished its binding affinity for as E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar, Alberdi E.M. A. Becerra S.P. Investig. Ophthalmol. Vis. Sci. 2001; 42: Scholar). the region of PEDF is for collagen used PEDF chemically modified with an form of which with as on of to of collagen I was in that of were in with collagen which increased with collagen I. In the bound PEDF was not detected with collagen I and with collagen I. a of the for collagen I of PEDF and in the of The modified a ionic to from collagen I the PEDF These that the to PEDF did not prevent binding to collagen increased In addition to excluding the region of the PEDF protein as site for interaction with collagen show that of its charged lysines increased its binding affinity for collagen I. the serpin loop of PEDF has a site S.P. A. P. Notario V. J. Biol. Chem. 1995; Scholar). PEDF and were by controlled proteolysis with and for collagen I that the binding was not abolished of the serpin loop of either PEDF or that region is for the collagen I the negatively charged of PEDF is directly involved in binding to collagen PEDF with as the in aspartic acid and glutamic acid were with of in the of a of containing as or to prevent The PEDF proteins as with of detected only with the of the the region of PEDF, the PEDF proteins were to heparin affinity that the proteins heparin-binding site with a heparin affinity as the that been that in the of of heparin affinity 3 and to in The modified PEDF proteins were for collagen I binding in with of in a of affinity for binding collagen I These that the aspartic and glutamic acid residues of PEDF were for its binding to collagen I and that they are directly involved in interactions with collagen I. The of region of PEDF for the binding to collagen I interactions at positively charged in collagen I. was used as a for the binding of the acidic region of PEDF to collagen I. binding were under ionic in which the interactions not E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar). that of heparin competed with PEDF for the binding to collagen I. These that PEDF a positively charged binding region in collagen I with heparin and that the binding site for collagen I in PEDF is formed by a negatively charged region on the surface of the In have and analyzed the interactions PEDF and collagen and for a collagen site on PEDF. show that the extracellular neurotrophic and antiangiogenic serpin PEDF to on collagens under conditions. when PEDF and collagen I are both in solution, when one is immobilized, or even when collagen I is denatured under reducing conditions. We present for ionic interactions a negatively charged in the and PEDF protein rich in glutamic acid and aspartic acid residues and positively charged in collagen I These interactions may in and may play in the of PEDF and/or in its biological e.g. antiangiogenic activities in the cornea and vitreous. PEDF has the affinity for collagen I collagens immobilized (10Kozaki K. Miyaishi O. Koiwai O. Yasui Y. Kashiwai A. Nishikawa Y. Shimizu S. Saga S. J. Biol. Chem. 1998; 273: 15125-15130Google and in solution, as collagens are in solution, the interactions have a binding nature as by the binding for collagen is immobilized, both the binding and in the affinity for the I interactions and as a for affinity. In surface revealed that interaction has and is In when the of D of the the is to This is to the binding in interactions in the range of to are with involved in cell Huang U. P.A. D. J. Biol. Chem. 2001; Scholar, P. J. Biol. Chem. 1998; 273: Scholar, C. Investig. Ophthalmol. Vis. Sci. 2001; 42: Scholar, P.A. J. Biol. Chem. 1998; 273: Scholar, J. Biol. Chem. 2002; Scholar). for a collagen site on PEDF. The affinity of PEDF for immobilized collagen I (K D = is distinct from its affinity for neurotrophic (K D = and (K D = for E. Alberdi E. Gettins P.G. Becerra S.P. Protein Sci. 1996; 5: 2575-2582Google Scholar, E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar, E. Becerra S.P. J. Biol. Chem. 1999; a distinct and binding site for collagen I on the PEDF protein In the of by the containing the PEDF site E. Becerra S.P. J. Biol. Chem. 1999; Scholar, Alberdi E.M. A. Becerra S.P. Investig. Ophthalmol. Vis. Sci. 2001; 42: is in with and binding for collagen I and the neurotrophic The that of lysines of the binding affinity for heparin E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar, Alberdi E.M. A. Becerra S.P. Investig. Ophthalmol. Vis. Sci. 2001; 42: not for collagen to distinct and binding for and three binding are of the serpin loop in PEDF. The of the affinity of PEDF for collagen I with an in ionic strength, a or of of PEDF, e.g. PEDF, is consistent with an charged region of PEDF as a binding site for collagen I. The of PEDF has distinct with ionic located from E. Hyde C.C. Becerra S.P. Biochemistry. 1998; 37: 10643-10652Google Scholar, M. Gettins P.G. K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: Scholar). The positively charged region is rich in acid lysines and with chains containing exposed to the surface of the protein, the negatively charged region is rich in aspartic and glutamic Glu42, Glu43, Asp44, Asp64, Asp256, Asp258, Glu290, Glu291, Glu296, Asp300, and with chains containing exposed to the surface of the protein the is the and the the acidic region of PEDF and collagen I the in that were at as the the of positively charged that are and the for the region is e.g. the of charged a in the negatively charged heparin Alberdi E.M. A. Becerra S.P. Investig. Ophthalmol. Vis. Sci. 2001; 42: and an in for collagen I when the acidic region of PEDF is PEDF, the of negatively charged a in the for positively charged areas in collagen I and an in for heparin that or of the aspartic and glutamic the acidic region of the PEDF protein are available to interact directly with positively charged areas in collagen I. In the of PEDF, acidic region, to the collagen site, is distinct and from both of the binding for the neurotrophic and heparin and from the serpin exposed loop for in the collagen I The heparin and PEDF for collagen I binding to binding for in collagen I. The binding affinity of heparin for collagen I (K D = Proc. Natl. Acad. Sci. U. S. A. 1998; is to that of that heparin and PEDF for collagen I. of in collagen I has that collagen I has positively charged areas that interact with J. L. J. Biol. Chem. 2002; Scholar). Several on collagen I have been to sulfate binding to type I collagen. of at of with one of the and is a of the These that the binding of sulfate and/or PEDF to collagen I which play a role in cell an in In triple helical of the of were found to in the collagen an for Proc. Natl. Acad. Sci. U. S. A. 1998; Scholar). In the in neovascularization or the of collagen, PEDF cell and on collagen. with known to to collagen I as and that the I interactions have binding to for biological activities of using surface with immobilized collagen I and as and J. Biol. Chem. 2002; found a D of an of and a (K of and J. Biol. Chem. 2001; the binding of and to bovine collagen I by and a collagen at (K of nm and a of However, the collagen binding in is to which with the collagen-binding region of J. Biol. Chem. 2002; Scholar, N. Adv. Exp. Med. Biol. 1997; 425: is not to the collagen-binding site for PEDF. In to PEDF, is an serpin and a role in collagen its is involved in of and cell C. Investig. Ophthalmol. Vis. Sci. 2001; 42: Scholar, M. Volpert O. Bouck N. Med. 2000; 6: Scholar). The collagen binding property of PEDF that PEDF cell interactions and has a as a cell molecule and/or the PEDF has binding for collagens and glycosaminoglycans, which are of cell The affinity of PEDF for extracellular matrix may even that for extracellular matrix as its affinity for collagen when the site of PEDF is and and even with the of the extracellular and of extracellular matrix e.g. collagen, glycosaminoglycans, as as of extracellular which development, with and in as and wound healing, (17Bishop P.N. Prog. Retin. Eye Res. 2000; 19: 323-344Google Scholar, L. Biol. 2001; Scholar, P. L. Scholar, J. P. L. N. Y. Acad. Sci. Scholar, D.W. J. 2001; Scholar, S. Investig. Ophthalmol. Vis. Sci. 1999; Scholar, I. D. M. Eye Res. 2000; Scholar, J. Biol. Chem. Scholar, J. 1998; may and the of the antiangiogenic activities of PEDF. We for with the and for protein and for with