Heparan Sulfate Mediates bFGF Transport through Basement Membrane by Diffusion with Rapid Reversible Binding

Basic fibroblast growth factor (bFGF) is a pluripotent cytokine with a wide range of target cells. Heparan sulfate binds bFGF, and this interaction has been demonstrated to protect bFGF against physical denaturation and protease degradation. The high concentrations of heparan sulfate in basement membranes have implicated these matrices as storage sites for bFGF in vivo. However, the mechanisms by which basement membranes modulate bFGF storage and release is unknown. To gain insight into these mechanisms, we have developed experimental and mathematical models of extracellular growth factor transport through basement membrane. Intact Descemet's membranes isolated from bovine corneas were mounted within customized diffusion cells and growth factor transport was measured under a variety of conditions that decoupled the diffusion process from the heparan sulfate binding phenomenon. Transport experiments were conducted with bFGF and interleukin 1β. In addition, bFGF-heparan sulfate binding was disrupted in diffusion studies with high ionic strength buffer and buffers containing protamine sulfate. Transport of bFGF was enhanced dramatically when heparan sulfate binding was inhibited. This process was modeled as a problem of diffusion with fast reversible binding. Experimental parameters were incorporated into a mathematical model and independent simulations were run that showed that the experimental data were accurately predicted by the mathematical model. Thus, this study indicated that basement membranes function as dynamic regulators of growth factor transport, allowing for rapid response to changing environmental conditions. The fundamental principles controlling bFGF transport through basement membrane that have been identified here might have applications in understanding how growth factor distribution is regulated throughout an organism during development and in the adult state. Basic fibroblast growth factor (bFGF) is a pluripotent cytokine with a wide range of target cells. Heparan sulfate binds bFGF, and this interaction has been demonstrated to protect bFGF against physical denaturation and protease degradation. The high concentrations of heparan sulfate in basement membranes have implicated these matrices as storage sites for bFGF in vivo. However, the mechanisms by which basement membranes modulate bFGF storage and release is unknown. To gain insight into these mechanisms, we have developed experimental and mathematical models of extracellular growth factor transport through basement membrane. Intact Descemet's membranes isolated from bovine corneas were mounted within customized diffusion cells and growth factor transport was measured under a variety of conditions that decoupled the diffusion process from the heparan sulfate binding phenomenon. Transport experiments were conducted with bFGF and interleukin 1β. In addition, bFGF-heparan sulfate binding was disrupted in diffusion studies with high ionic strength buffer and buffers containing protamine sulfate. Transport of bFGF was enhanced dramatically when heparan sulfate binding was inhibited. This process was modeled as a problem of diffusion with fast reversible binding. Experimental parameters were incorporated into a mathematical model and independent simulations were run that showed that the experimental data were accurately predicted by the mathematical model. Thus, this study indicated that basement membranes function as dynamic regulators of growth factor transport, allowing for rapid response to changing environmental conditions. The fundamental principles controlling bFGF transport through basement membrane that have been identified here might have applications in understanding how growth factor distribution is regulated throughout an organism during development and in the adult state. Basic fibroblast growth factor (bFGF) 1The abbreviations bFGFbasic fibroblast growth factorHSheparan sulfateECMextracellular matrixDMDescemet's membraneBSAbovine serum albuminPBSphosphate-buffered salineIL-1βinterleukin 1β 1The abbreviations bFGFbasic fibroblast growth factorHSheparan sulfateECMextracellular matrixDMDescemet's membraneBSAbovine serum albuminPBSphosphate-buffered salineIL-1βinterleukin 1βis a pluripotent growth factor that affects a wide range of cell types of mesodermal, endodermal, and ectodermal origin. It has been implicated in processes ranging from wound healing to tumor growth (1Baird A. Walicke P.A. Br. Med. Bull. 1989; 45: 438-452Crossref PubMed Scopus (219) Google Scholar, 2Bikfalvi A. Klein S. Pintucci G. Rifkin D.B. Endocr. Rev. 1997; 18: 26-45Crossref PubMed Scopus (848) Google Scholar). Like the other members of the FGF family, bFGF binds heparin and heparan sulfate (HS). The association of bFGF with heparin/HS is characterized by a dissociation constant in the nanomolar range. This interaction stabilizes the growth factor and protects it from proteolytic degradation (3Saksela O. Moscatelli D. Sommer A. Rifkin D.B. J. Cell Biol. 1988; 107: 743-751Crossref PubMed Scopus (651) Google Scholar). In vivo, HS is found linked to a protein core as a heparan sulfate proteoglycan. Basic FGF has been localized to HS sites in a specific extracellular matrix (ECM), the basement membrane, and it is a potent mitogen for the endothelial cells that border basement membranes (4Folkman J. Klagsbrun M. Sasse J. Wadzinski M. Ingber D. Vlodavsky I. Am. J. Pathol. 1988; 130: 393-400PubMed Google Scholar, 5Jeanny J. Fayein N. Moenner M. Chevallier B. Barritault D. Courtois Y. Exp. Cell Res. 1987; 171: 63-75Crossref PubMed Scopus (106) Google Scholar). However, under normal physiological conditions these cell layers remain relatively quiescent. This observation, in conjunction with other in vivo and in vitro evidence, suggests that basement membrane HS plays a critical role in modulating the activity of bFGF by providing a natural reservoir for bFGF (6Baird A. Ling N. Biochem. Biophys. Res. Commun. 1987; 142: 428-435Crossref PubMed Scopus (310) Google Scholar, 7Vlodavsky I. Folkman J. Sullivan R. Fridman R. Ishai-Michaeli R. Sasse J. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2292-2296Crossref PubMed Scopus (840) Google Scholar, 8Flaumenhaft R. Moscatelli D. Saksela O. Rifkin D.B. J. Cell. 1989; PubMed Scopus Google Scholar, M. M. G. J. Cell. 1989; PubMed Scopus Google Scholar, S. Klagsbrun M. Folkman J. Vlodavsky I. 1989; PubMed Scopus Google Scholar). However, it is how the basement membrane HS as a reservoir and the transport of bFGF through fibroblast growth factor heparan sulfate extracellular matrix Descemet's membrane bovine serum interleukin 1β fibroblast growth factor heparan sulfate extracellular matrix Descemet's membrane bovine serum interleukin 1β mechanisms have been for the of bFGF by degradation of HS the membrane by protease the growth factor of with the HS and a of diffusion the HS allowing for through the This of process might that the HS concentrations a a HS through the matrix is is that the rapid of bFGF association and dissociation from HS a dynamic reservoir of bFGF that release bFGF in response to in the bFGF HS studies the of binding that the of an is PubMed Scopus Google Scholar). Thus, the rapid binding a dynamic storage and release for bFGF that matrix degradation to in vitro have the of bFGF through extracellular matrices and and Courtois I. Courtois Y. J. Cell. PubMed Scopus Google conducted diffusion experiments with bFGF through an of tumor matrix that is in In the of bFGF heparin with bFGF, the of bFGF that a of R. Moscatelli D. Rifkin D.B. J. Cell Biol. PubMed Scopus Google showed that as and bFGF demonstrated diffusion through and that heparin and protamine sulfate the of diffusion of bFGF and for diffusion within and cell a study the of bFGF demonstrated with denaturation of bFGF with A. D. Res. Scopus Google Scholar). It is that denaturation of bFGF in a in heparin binding activity which might have to these critical studies identified heparan sulfate as an factor that the of bFGF through the of this matrix In addition, data the of bFGF through basement membrane experimental to developed basement membrane to and to how in the of bFGF, transport in basement critical in the process identified by mathematical models that the the matrix and a of the to In this a for understanding the of factor parameters In the study we the transport of bFGF Descemet's membrane the basement membrane of the a model that the diffusion of bFGF through the of the membrane with reversible association of bFGF to HS Descemet's membrane is in the of the the and the is by a of R. PubMed Scopus Google Scholar, Cell Res. PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar). The of the other basement membrane and heparan sulfate has been demonstrated Google Scholar, U. Exp. Res. PubMed Scopus Google Scholar). The of the has been by to a of by and in Cell Res. PubMed Scopus Google Scholar). as as it to it for diffusion the was the basement membrane identified as a vivo bFGF reservoir (4Folkman J. Klagsbrun M. Sasse J. Wadzinski M. Ingber D. Vlodavsky I. Am. J. Pathol. 1988; 130: 393-400PubMed Google Scholar). Basic FGF diffusion through the was measured with a diffusion under conditions that decoupled the diffusion process from the binding phenomenon. The data was incorporated into a model with independent of the This model was to experimental and suggests that the of the basement membrane bFGF reservoir by diffusion and binding of bFGF was from 1β were from was a of the PubMed Scopus Google has been demonstrated to as as bFGF in and binding to cells PubMed Scopus Google Scholar, J. Biol. PubMed Google Scholar, M. J. Biol. PubMed Scopus Google Scholar). and were from to bFGF was from was from serum was from was from were from U. 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Biol. PubMed Scopus Google Scholar). The membranes were in in with for with and with The were with the been a of was the and to for to bFGF was to heparin binding activity as by Descemet's membranes were as and the were U. S. were in and were to and to in for The of the membranes was by the for to and by the of containing binding were to the membranes in the and with constant for The was The membrane containing and were and the was measured with a The of was by was through the membrane containing a of and were in these was measured in a The was from the membrane the of from the membranes the of and the of the The for bFGF in the was and for was The the range for The of this study was to the process of bFGF transport through basement membrane with a model. Thus, bFGF through the matrix of the basement membrane and reversible association with the HS with by the is the of bFGF in the membrane. 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To gain insight into the role of bFGF heparin binding in controlling transport, we conducted transport studies with the binding bFGF that was isolated from the during This of protein that to heparin as a of the process from the in a of bFGF that was the and was that it The transport of and Descemet's membrane were the of these studies bFGF that to heparin was to the membrane with The of the binding of bFGF, for membrane and bFGF was the of The bFGF by the bFGF The diffusion of bFGF that heparin was dramatically with the a an of was for the bFGF diffusion for the bFGF was the diffusion from diffusion of the diffusion data in was conducted as under the from the of a of was by a The of the and diffusion was by the by the of that membrane and by the by the measured in the was the measured of for and were from and the range of experiments conducted under the indicated in a of the diffusion data in was conducted as under the from the of a of was by a The of the and diffusion was by the by the of that membrane and by the by the measured in the was the measured of for and were from and the range of experiments conducted under the indicated In the diffusion bFGF was in the by the of the were experiments to that the protein accurately the of identified a protein to that of the bFGF that the was the protein containing Thus, in the accurately the of To that HS was in modulating bFGF through membranes were with was this the within heparan sulfate that have been implicated in bFGF binding S. R. R. Rev. Biochem. Biol. PubMed Scopus Google Scholar). studies with these membranes and in a in bFGF transport and the from a of this study indicated that the HS bFGF Thus, the of the that HS is an of bFGF transport, were to the HS binding from the transport process in to for the were to a for the and to the of bFGF interaction within and through the The diffusion of bFGF was with that of is a cytokine of with a of it with bFGF, the of is to bFGF, bFGF and J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). from and that the have J. Biol. PubMed Scopus Google Scholar, R. PubMed Scopus Google with bFGF a of and a of we that the have was as a bFGF that demonstrated transport to the binding bFGF and this data and the for an for of was the association of bFGF with heparan sulfate is the binding is from the diffusion and the of bFGF to measured for bFGF and ionic strength in the membrane, and ionic for high concentrations bFGF binding to HS D. J. Cell. 1987; PubMed Scopus Google Scholar, Ishai-Michaeli R. R. Vlodavsky I. J. Biol. PubMed Scopus Google Scholar). The diffusion of bFGF and through Descemet's membrane was conducted in in a high the of bFGF through the membrane was dramatically as with bFGF in a physiological ionic strength by bFGF from with the HS in the The of bFGF and in were However, the for in a buffer were of the of in physiological In an independent the was to in in The in into a Cell Res. PubMed Scopus Google and in The that the high the HS from the matrix during the was Descemet's membranes were in and the was This was against with and for heparan sulfate by and the binding J. Cell. 1997; PubMed Scopus Google Scholar). HS was in the the of of the heparan sulfate and the of HS within the we that of HS was by sulfate binds heparan the association of bFGF with the HS G. D. J. Cell. 1987; PubMed Scopus Google Scholar). protamine sulfate was to bFGF binding from diffusion the of the membrane. were with protamine sulfate for to that the HS sites in the membrane were and the diffusion studies were conducted in this of protamine sulfate. an of the protamine sulfate was the of The of protamine sulfate in a in bFGF as with the of bFGF in The of bFGF with protamine sulfate was within of the of The of bFGF from this data was The for bFGF with protamine sulfate and diffusion were as have and in the protamine sulfate bFGF was by the HS in the the data the interaction as the of through the membrane. experiments with the concentrations of bFGF in to bFGF to HS sites in the membrane and the bFGF to the measured when the interaction was However, experiments that denaturation of bFGF within the diffusion and experiments The to concentrations of Thus, transport was a of bFGF a of during which the HS sites were the to of that from experiments with bFGF and protamine sulfate In to the experimental by the were measured and incorporated into a of the The mathematical model the of the interaction and the of Thus, HS was from the by and the HS was by and and The of the HS was to the and the the of HS in was to a range of bFGF binding to HS was conducted a rapid to and the of bFGF to this of the interaction of bFGF with HS was to with a of of bFGF of HS J. and M. A. The binding were to and of and The from these and the measured that binding diffusion a and bFGF within the membrane. parameters were into the mathematical model and experiments were The from independent that were in the mathematical model in in model of bFGF transport through the of the is In the mathematical the of sites was as the of the parameters in this were measured in independent experiments were through the binding constant the of the is In the mathematical the of sites was as the of in a the parameters in this were measured in independent experiments were through the binding constant the of bFGF as in diffusion experiments through Descemet's membrane the simulations were conducted a range of HS concentrations that the measured and the of the the of HS that we measured in the the model predicted a of by a constant that was within of the experimental the range of HS concentrations the the predicted from to within the of the model the simulations predicted transport data that were to the experimental and that the transport of bFGF a with a model. The of growth and as a to cell function and has as an throughout Cell. PubMed Scopus Google Scholar, N. Biol. 1997; PubMed Scopus Google Scholar, A. D. PubMed Scopus Google Scholar, Cell Biol. PubMed Scopus Google Scholar, S. Y. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, M. Google Scholar). However, understanding of how within and adult has been that the extracellular matrix might in N. Biol. 1997; PubMed Scopus Google Scholar, Cell Biol. PubMed Scopus Google Scholar, PubMed Google Scholar, J. O. J. 1997; PubMed Scopus Google Scholar). In the binding of growth as members of the FGF and growth factor with extracellular matrix has been to a critical in cell and J. O. J. 1997; PubMed Scopus Google Scholar). In the study we have the role of the extracellular matrix as a of growth factor transport a fundamental a model of bFGF transport through an extracellular Descemet's membrane, we have identified diffusion with rapid reversible binding to heparan sulfate as the critical that bFGF we have and mathematical models of bFGF that to that to the transport of bFGF through the extracellular matrix is a we a model of diffusion with reversible binding to matrix sites to this conducted experiments the as a model to of the of bFGF under conditions in which binding of bFGF to HS in the matrix was and were measured in The from these as as of membrane HS bFGF binding and were incorporated into a of the diffusion experiments were conducted to these processes accurately The experimental were by the model and Thus, that reversible binding of bFGF to heparan sulfate a for to the of bFGF in vivo that degradation of the However, in studies bFGF binding was we an of bFGF data that degradation of matrix heparan sulfate dramatically bFGF release experimental to models the of bFGF to HS bFGF through the for bFGF by HS might concentrations of HS we in the might in the extracellular studies bFGF transport through extracellular matrix have identified the interaction with heparin as critical to this In a study by and Courtois I. Courtois Y. J. Cell. PubMed Scopus Google transport of bFGF a basement membrane enhanced by the bFGF by that binding to heparan sulfate sites to bFGF and that these sites with bFGF the bFGF with heparin the study is with the of the experimental by and Courtois and the that parameters membrane HS were and to the by R. Moscatelli D. Rifkin D.B. J. Cell Biol. PubMed Scopus Google that binding of bFGF to sites within an matrix diffusion In this bFGF diffusion in was enhanced in the of that these were ionic of bFGF with sites in the these studies a diffusion with binding model for bFGF through In the study we have these by this process in to a model of this transport The experimental we isolated of to the diffusion of other through the basement membrane. this is the of an experimental that of transport through basement membrane the of in Descemet's membrane for The of through with and Descemet's membrane was measured M. D. Exp. Res. PubMed Scopus Google and a for the of for membranes was we measured of this to a of for in the of the this is in with the measured here for bFGF and Thus, data with experimental is to a of basement membrane transport within the of an for a protein of in to the a bFGF of the is This that the of the a transport to in the of binding. It is that the of the and by the of protein and within the membrane in protein diffusion Am. J. 1989; Scopus Google Scholar). 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However, in this a membrane was with In the this membrane is to and the of bFGF from the membrane was The of bFGF the of the membrane is as a function of the bFGF has been from the membrane. The of bFGF through membranes was an In an the basement membrane is by a cell and the other by The cell and layers to heparan sulfate as bFGF from a basement membrane to by the of the membrane. that from the is that basement membranes dynamic for in the basement membrane, the bFGF is from protease activity and by interaction with heparan sulfate D. J. J. Cell. PubMed Scopus Google Scholar, A. Rifkin D. J. Cell. 1989; PubMed Scopus Google Scholar, S. Klagsbrun M. R. M. A. Vlodavsky I. J. Cell Biol. 1989; PubMed Scopus Google Scholar). However, in the of the basement membrane, as physical in rapid release of we and have identified a of bFGF binding and release from extracellular matrix is a critical to cell and response to Am. J. PubMed Scopus Google Scholar, Am. J. PubMed Scopus Google Scholar). the of cell J. Cell. 1997; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google of in J. Biol. PubMed Scopus Google and wound healing J. PubMed Scopus Google Scholar). The process of bFGF transport through the extracellular matrix with a model bFGF diffusion with reversible binding to the HS of the matrices critical in in in normal and in the of in controlling extracellular is an understanding of and tumor This study has demonstrated that a mathematical model that the and of bFGF diffusion with association to HS the of the process of bFGF transport basement membrane. from for and providing protein to in this for critical of the to for throughout this