 |
INTRODUCTION |
Basic fibroblast growth factor
(bFGF)1 is 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 (1, 2). 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 (3). 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 (4, 5). 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 (6-10). However, it is unclear how the basement membrane HS functions as a reservoir and regulates the transport of
bFGF through ECM.
Several mechanisms have been proposed for the ECM-bFGF reservoir.
Release of bFGF could be triggered by degradation of HS or the membrane
by glycosaminoglycan degrading enzymes or protease activity.
Alternatively, the growth factor could take advantage of nonspecific
interactions with the HS chains and experience a form of
one-dimensional diffusion along the HS chains allowing for accelerated
movement through the matrix. This type of process might require that
the HS concentrations reach a level where a continuous HS pathway
through the matrix is available. Another possibility is that the rapid
kinetics of bFGF association and dissociation from HS could provide a
dynamic reservoir of bFGF that could release or incorporate bFGF in
response to changes in the local bFGF or HS concentrations. Our
previous studies on the kinetics of bFGF·HS binding suggest that the
average lifetime of an bFGF·HS complex is approximately 1 min (11).
Thus, the rapid binding could facilitate a dynamic storage and release
system for bFGF that would not absolutely require matrix degradation to
trigger release.
Researchers using in vitro systems have studied the
properties of bFGF movement through artificial extracellular matrices and negatively charged hydrogels. Dabin and Courtois (12) conducted diffusion experiments with radiolabeled bFGF through
MatrigelTM, an extract of Engelbreth Holm Swarm tumor
matrix that is rich in HS. In their studies, either increasing the
concentration of bFGF or adding soluble heparin along with bFGF,
increased the amount of bFGF that crossed a layer of matrigel.
Likewise, Flaumenhaft et al. (13) showed that positively
charged proteins such as cytochrome c and bFGF demonstrated
reduced diffusion through negatively charged agarose gels, and that
both heparin and protamine sulfate can increase the radius of diffusion
of bFGF and cytochrome c. They reported similar phenomena
for diffusion within fibrin gels and cultured cell layers. Furthermore,
a recent study on the permeation of bFGF across rabbit buccal mucosa
demonstrated increased permeation with denaturation of bFGF with
guanidine HCl (14). It is likely that denaturation of bFGF resulted in
a loss in heparin binding activity which might have imparted increased
permeation properties to bFGF.
While these critical studies identified heparan sulfate as an important
factor that can control the movement of bFGF through matrices, they did
not provide information about the mechanism of this matrix transport.
In addition, no data currently exist on the movement of bFGF through
actual basement membrane samples. More controlled experimental systems
need to be developed using actual basement membrane samples to obtain
mechanistic information, and to determine how changes in the nature of
bFGF, or its concentration, would affect transport in basement
membranes. Furthermore, critical elements in the process can be
identified by building mathematical models that incorporate the
potentially important interactions between the matrix and its soluble
components, thus enabling a comparison of the model's predictions to
empirical studies. In this way, a systematic framework for
understanding the interplay of key matrix/growth factor parameters can
be developed.
In the present study we analyzed the transport of bFGF across
Descemet's membrane (DM), the basement membrane of the corneal endothelium, using a model that considers the diffusion of bFGF through
the interstices of the membrane coupled with fast, reversible association of bFGF to resident HS chains. Descemet's membrane is
situated in the anterior region of the cornea between the corneal stroma and the endothelium. Its molecular structure is dominated by a
dense meshwork of collagen VIII (15-17). The presence of the other
major basement membrane components, collagen IV, laminin, entactin,
fibronectin, and perlecan heparan sulfate proteoglycan has also been
demonstrated (18, 19). The ultrastructure of the DM has been determined
by electron microscopy to be based on a lamellar hexagonal lattice of
80 nm nodes connected by rods approximately 120 nm long and 25 nm in
diameter (16). Bovine DM can be as much as 100-µm thick making it
possible to physically isolate it for diffusion studies. Furthermore,
the DM was the first basement membrane identified as a potential
in vivo bFGF reservoir (4).
Basic FGF diffusion through the DM was measured with a diffusion
chamber apparatus under different conditions that decoupled the
diffusion process from the binding phenomenon. The diffusivity data was
incorporated into a diffusion/binding model along with independent
measurements of the bFGF-HS interaction. This model was used to
simulate experimental results and suggests that the essential elements
of the basement membrane bFGF reservoir are governed by diffusion and
binding of bFGF.
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EXPERIMENTAL PROCEDURES |
Materials--
bFGF (human recombinant) (18 kDa) was from
Scios-Nova (Mountain View, CA). Human recombinant
125I-interleukin 1
(IL-1
) and
125I-Bolton-Hunter reagent were obtained from NEN Life
Science Products Inc. (Boston, MA). 125I-bFGF was prepared
using a modification of the Bolton-Hunter method (11).
125I-bFGF has been demonstrated to be as active as
unlabeled bFGF at stimulating DNA synthesis in and binding to quiescent
Balb/c3T3 cells (11, 20, 21). Sodium chloride, sodium azide, pepstatin, leupeptin, N-ethylmaleimide, trichloroacetic acid, and
potassium iodide were purchased from Sigma. Monoclonal antibody to
human recombinant bFGF (mouse, anti-human) was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Horseradish peroxidase-linked anti-mouse IgG (from sheep) whole antibody was from Amersham. Bovine
serum albumin (BSA) (Bovuminar Cohn Fraction V) was purchased from
Intergen (Purchase, NY). Dulbecco's phosphate-buffered saline (PBS)
was obtained from Life Technologies, Inc. (Grand Island, NY).
Microreaction columns were obtained from U. S. Biochemical Corp.
(Cleveland, OH). Sephadex G-25 PD-10 columns and heparin-Sepharose affinity chromatography media were from Pharmacia (Uppsala, Sweden). Diffusion chambers and clamps were obtained from Crown Glass Co. (Sommerville, NJ). Solution concentrations of bFGF were measured, in
some cases, using a human bFGF Quantikine Immunoassay (R&D Systems,
Minneapolis, MN). Tween 20 was purchased from Bio-Rad. 3-Glycidoxypropyltrimethoxysilane was obtained from Aldrich (Milwaukee, WI). Immobilon P and Duropore 0.65-µm membranes were obtained from
Millipore (Bedford, MA).
Membrane Dissection and Storage--
Whole bovine eyes were
obtained from Pel-Freeze (Rodgers, AK) and shipped overnight on ice
after harvest. The cornea was removed by cutting along its periphery
with curved scissors, and placed endothelium face up on a spherical
surface. The endothelium was removed by repeated wiping with a tissue
followed with PBS rinses. A curved Teflon spatula was used to score the
surface of the DM into quarters, and the DM was wetted with PBS. The
exposed edge of the DM was gradually teased away from the stroma using
the wedge-shaped edge of the Teflon spatula. Upon removal, the DM was
placed in a vial with storage buffer (PBS, 0.1% sodium azide, 1 µg/ml pepstatin, 0.5 µg/ml leupeptin, and 1 mM
N-ethylmaleimide). Membrane sections were stored in the
refrigerator for as long as 2 months with no noticeable change in
transport properties, but they were generally used within 3 weeks of
dissection. In control studies, the permeability of freshly isolated
membranes was analyzed over 4 days using a mixture of fluorescent
dextrans and no time-dependent changes were noted.
Furthermore, the permeability of bFGF was measured under identical
experimental conditions in membranes stored for 5, 14, 27, and 36 days.
In these studies FGF permeability varied by
13% from experiment to
experiment with neither an increasing nor decreasing trend with time of
membrane storage. The variability in bFGF permeability between
experiments was generally less than or equal to that observed with
multiple membranes in a single experiment.
Histology--
Dissected membranes were fixed in formaldehyde,
embedded in paraffin, and stained for basement membrane with the
periodic acid shiff reagent (22). Visual inspection of membrane
preparations revealed no evidence of residual corneal stroma or
endothelium (data not shown).
bFGF Purification on Heparin-Sepharose--
Immediately prior to
each diffusion experiment, 125I-bFGF samples were purified
on heparin columns to separate non-heparin binding bFGF and dissociated
free 125I from native 125I-bFGF.
Heparin-Sepharose CL-6B was dissolved in deionized water to form a 1:2
gel to water slurry. A 100-µl column was poured using U. S. Biochemical Corp. compact reaction columns and 1 liter of PBS was run
over the column overnight. Leaching rates of heparin from the column
after this wash were shown to be negligible. The column was
equilibrated with approximately 30 ml of chilled PBS, 1 mg/ml BSA
(PBS-BSA) buffer. All buffers that contacted bFGF were prepared with
PBS with 1 mg/ml BSA as a carrier protein to reduce surface adsorption
losses of bFGF. A 100-µl aliquot of 125I-bFGF (1-3
µg/ml) diluted with 200 µl of PBS-BSA was applied to the column.
The column with bFGF was maintained in suspension on ice for a 10-min
incubation period to allow bFGF to bind the heparin. 30 ml of chilled
0.5 M NaCl PBS were passed over the column to remove free
label and unbound bFGF. After draining the column to its bed surface,
300 µl of 3.0 M NaCl in 1 mg/ml PBS was added to the gel,
and incubated on ice for another 10 min. The column was centrifuged
(10,000 × g for 30 s) into a 2-ml centrifuged tube to remove the eluant. In experiments conducted in 3.0 M NaCl buffer, the centrifugation step was omitted, and
bFGF was eluted with 4 × 200-µl volumes of 3.0 M
NaCl. The final concentration of 125I-bFGF was determined
by trichloroacetic acid precipitation. The radioactivity in the
heparin-Sepharose purified material was greater than 99.0%
precipitable by trichloroacetic acid.
Purification of Non-heparin Binding bFGF and IL-1
--
Size
exclusion gel filtration chromatography was used to separate free
125I-label from 125I-IL-1
and inactive
125I-bFGF. In these cases, a PD-10 column containing
Sephadex G-25 gel filtration media was used to remove the free label.
The column was equilibrated and the separation was conducted in PBS-BSA
buffer. The radioactive peak that eluted in the void volume was
collected as the radioactive protein. Greater than 98.5% of the
radioactivity in the void volume was trichloroacetic acid precipitable.
Additional gel filtration chromatographic analysis of the non-binding
bFGF showed no evidence of oligomerization as compared with native bFGF. Thus, the inactive bFGF fraction appears to be similar in size
yet without the heparin-binding affinity of native bFGF.
Diffusion Experiments--
Fig. 1
is a schematic of the diffusion cell apparatus with membrane slide
mounts. The chamber apparatus was secured with a metal clamp attached
to a Lucite base. The glass diffusion cells and mounts were coated to
reduce protein adsorption through the covalent attachment of short
carbohydrate chains to their glass surfaces (23). The chambers and
membrane mounts were soaked in 6 N HCl overnight and washed
exhaustively with deionized water to remove impurities. The coating
solution was prepared with 25 ml of 3-glycidoxypropyltrimethoxysilane,
225 ml of deionized water, and 62.5 µl of 1 M HCl to
adjust the solution pH to 3.5. The chambers and slides were coated for
6 h at 90 °C, including a 1-h warm-up period to prevent the
glass chambers from fracturing. Following the coating the chambers were
rinsed thoroughly with deionized water and placed in a dry box for
storage. Control experiments using model compounds (urea and sucrose),
with known aqueous diffusion coefficients, and a synthetic hydrophilic
membrane (Durapore 0.65 µm) were conducted to ensure that the
membrane slide mounts did not introduce significant boundary layer
effects near the surface of the membranes.

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Fig. 1.
Diffusion chamber apparatus. The
diffusion chamber apparatus consisted of a portion of Descemet's
membrane secured with a pair of glass slide mounts. The slide mounts
were placed between the two diffusion chambers. Both chambers were
mixed with 3 × 10-mm stir bars. Samples ports provided access to
each chamber.
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Membranes were placed between the two slide mounts, bolted in place,
and the slides were positioned between the two diffusion cells. A bead
of vacuum grease (Dow Corning Silicone vacuum grease) was applied
around the face of each of the diffusion cells to maintain a
water-tight seal. The clamp was tightened only until resistance was
felt so as not to puncture the membranes. 3 ml of 0.22-µm filtered
PBS-BSA was added to each chamber and the apparatus was placed on a
stir plate at 4 °C. A coated Teflon microstir bar was added to each
cell. These stir bars were surface modified by Corning Inc. with a
polyethylene oxide-like, non-ionic, hydrophilic coating to limit
protein adsorption to the Teflon (Corning, Inc., Corning, NY). The
stirring rate was adjusted to 500 rpm. The assembled chambers were
allowed to equilibrate for 15-20 h prior to initiating diffusion experiments.
The buffer in the sink chamber was removed and replaced with fresh
buffer immediately prior to the start of the experiment. The contents
of the source chamber was replaced with a phosphate buffer (10 mM NaPO4 pH 7.4) lacking salt in experiments
with native bFGF, since the 125I-bFGF sample provided
enough salt to bring the final concentration in the source chamber to
150 mM. Standard PBS-BSA was used in the source chamber in
studies with non-heparin binding bFGF or IL-1
. Experiments were
initiated by introducing purified radiolabeled protein to the source
chamber. Over the course of the experiment, 200-µl samples were
removed from the sink chamber at each time point. This volume was
replaced with fresh PBS-BSA. The source chamber was also sampled at
various time points during the course of the experiment. All samples
were subjected to trichloroacetic acid precipitation to quantify
125I-protein.
Trichloroacetic Acid Precipitation--
Trichloroacetic acid
precipitation was used to distinguish free radiolabel from radiolabeled
protein. For a 200-µl sample to be precipitated, the following
volumes were added: 1) 1.25 ml of PBS or saline; 2) 100 µl of 10 mg/ml BSA; and 3) 200 µl of 10 mM KI. The samples were
precipitated with 250 µl of 100% trichloroacetic acid, vortexed
vigorously, and placed on ice for 10 min. The samples were
centrifuged for 10 min at 1,800 × g and the
supernatant was removed. The pellets were washed with 500 µl of 14%
trichloroacetic acid, and centrifuged again. Radioactivity in the
pellet, supernatant, and wash solutions was measured in a
-counter
(Packard Auto-
5650).
Measuring Membrane Thickness--
The thickness of the membrane
is a critical parameter in the diffusion process. The thickness of each
membrane was measured after each diffusion experiment by taking
advantage of its natural tendency to curl over on itself. The membrane
was viewed in cross-section and photomicrographs were made at × 200 using a Nikon Diaphott microscope (Nikon). Membrane thicknesses
were measured with a calibrated micrometer photographed at the same
magnification. Based on 26 individual diffusion experiments with
Descemet's membrane, the average membrane thickness was 40 µm with a
standard deviation of 8 µm. The total thickness variation across a
given membrane averaged 14 µm. The data from each diffusion study was
normalized to the thickness of the particular membrane used. While
experiments conducted in 3.0 M NaCl buffer were also
normalized to the thickness of the particular membrane used in those
experiments, theoretical considerations suggest that membranes might be
altered physically by the high ionic strength. Thus to evaluate whether
the high salt conditions were altering membrane thickness, individual
membranes were fixed in place in a continuous flow viewing chamber
constructed of two glass slides and a neoprene gasket. Membranes within
chambers containing 150 mM NaCl PBS were positioned on a
microscope and photographed for baseline thickness measurements and
then the buffer was exchanged to 3.0 M NaCl phosphate
buffer through 20-gauge needle input and export ports within the
chamber. Ionic strength was monitored in the outflow with an on-line
conductivity meter and flow was stopped when the buffer was fully
exchanged. Membrane thickness measurements were taken at the exact same
positions along the fixed membrane over a 24-h period. An ~10%
reduction in membrane thickness was noted after membrane equilibration
into 3.0 M NaCl phosphate buffer.
Western Blotting and Autoradiography of Sink Cell bFGF--
At
the end (24 h) of diffusion experiments using high bFGF concentrations
(1 µM) samples were removed from the sink chambers and
subjected to SDS-polyacrylamide gel electrophoresis (15% running gel)
under reducing conditions (24). Control lanes were included containing
PBS-BSA buffer alone. Proteins were electrotransferred to Immobilon P
membranes and bFGF was detected with an anti-bFGF antibody (25). The
membranes were blocked overnight in 10% milk in PBS with 0.1% Tween
20, probed for 1 h at 37 °C with mAb anti-bFGF type II at
1:1000 dilution, and incubated with horseradish peroxidase-linked anti-mouse IgG (from sheep) at 1:1000 dilution. The bands were visualized with Renaissance Western blot chemiluminescent reagent (NEN
Life Science Products Inc.) on X-Omat AR scientific imaging film
(Kodak, Rochester, NY). After the chemiluminescent reagent had been
exhausted, a fresh piece of film was placed over the blot and allowed
to expose for 2 months at
70 °C to detect radioactive samples.
Sink chamber bFGF was also determined to retain its heparin binding
activity as determined by heparin-Sepharose chromatography.
Measuring Partition Coefficients--
Descemet's membranes were
dissected as described above and diced until the larger pieces were
approximately 1 mm2. U. S. Biochemical Corp.
microcolumns were incubated in PBS-BSA and then weighed dry. Membranes
(0.052-0.065 g) were added to each column and allowed to incubate in
PBS-BSA while being constantly inverted at 4 °C for 12 h. The
wet mass of the membranes was determined by centrifuging the columns
for 30 s at 50 × g to remove excess solution and
subsequently by measuring the total mass. Five hundred microliters of
PBS-BSA containing non-heparin binding 125I-bFGF or
125I-IL-1
were added to the membranes in the columns and
incubated at 4 °C with constant rotation for 5 h. The
centrifugation step was repeated. The membrane containing columns and
their incubation solutions were weighed and the radioactivity was
measured with a
-counter. The volume of incubation solution was
determined by mass difference. PBS-BSA was applied through the membrane
containing columns at 4 °C at a flow rate of 1 ml/min and 2-ml
fractions were collected. Radioactivity in these fractions was measured in a
-counter. The partition coefficient was determined from the
known membrane volume (Vmem), the total amount
of elutable radioactivity from the membranes (
mem), the
volume of incubation solution (Vbulk), and the
total cpm of the incubation solution (
bulk).
|
(Eq. 1)
|
The partition coefficient for bFGF in the DM was 1.48 ± 0.03 and for IL-1
was 1.49 ± 0.05. The reported errors
represent half the total range for duplicate determinations.
Diffusion/Binding Analysis--
The goal of this study was to
determine if the complex process of bFGF transport through basement
membrane could be described with a simple diffusion/binding model.
Thus, bFGF would diffuse through the matrix of the basement membrane
and experience rapid, reversible association with the resident HS
chains. Diffusion with reaction can be represented by the following
equation,
|
(Eq. 2)
|
Here, b is the molar concentration of free bFGF in
the membrane. The concentration of bFGF·HS complex is represented by
c. As this is a one-dimensional equation for diffusion,
t is time, and
is the distance through the matrix.
Deff is the effective diffusivity of bFGF in the
ECM. The diffusivity of bFGF through the tight meshwork of ECM is
expected to be much lower than the free aqueous diffusivity of bFGF.
K is a partition coefficient that relates the concentration
of unbound bFGF in the matrix to its bulk concentration. This value
will be influenced by the nature of bFGF's interaction with the
membrane and the proportion of the total aqueous volume of the matrix
that is available to bFGF. In this formulation of the problem, the
partition coefficient does not encompass the equilibrium binding
interaction of bFGF with HS.
The association of bFGF with HS sites in the membrane was modeled as a
reversible association with a single class of binding sites with no
cooperativity.
|
(Eq. 3)
|
where kon and koff
are the on- and off-rate constants for the interaction of bFGF with HS
and h is the concentration of uncomplexed HS sites in the
membrane. The concentrations of complex and free HS sites are also
subject to the following constraint,
|
(Eq. 4)
|
where htot is the total number of HS
sites. Values for both the concentration of complexes (c)
and the total concentration of HS sites (htot)
reflect the presence of multiple bFGF binding sites per HS molecule.
If the binding events take place on a much shorter time scale than
diffusion, then the free bFGF and HS will be at local equilibrium with
bound bFGF·HS complexes at each point in the membrane. This condition
is satisfied when the Damkholer numbers for binding and release are
much greater than 1.
|
(Eq. 5)
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|
(Eq. 6)
|
Under these circumstances, the equilibrium dissociation constant
(Kd) is sufficient to describe the reaction,
|
(Eq. 7)
|
At steady-state both time derivatives approach 0 in Equation 2,
and the flux through the membrane becomes a constant.
|
(Eq. 8)
|
Here, l is the membrane thickness. In our diffusion
experiments, where radiolabeled proteins moved from a source chamber of
high concentration to a sink chamber of much lower concentration,
C was effectively the source concentration. Membrane
thicknesses and the partition coefficient were measured directly. With
these values, Deff was determined experimentally
from the slope of a plot of flux versus time.
The assumptions outlined above are true even in the presence of a
reversible binding event in the membrane. Once the binding and release
have reached a steady state throughout the membrane, the flux through
the membrane becomes independent of the binding interaction. Thus, the
relevant parameters are the effective diffusivity, the partition
coefficient, the equilibrium binding constant, and the concentration of
binding sites.
Numerical Solution to Diffusion Reaction Problem--
Equations
2-4 were solved with a semi-implicit finite difference method (26)
using the following boundary and initial conditions,
|
(Eq. 9)
|
|
(Eq. 10)
|
Where A is the area available to diffusion,
b° is the initial concentration of bFGF in the source
chamber, V is the sink chamber volume, and the other
variables are as described above.
The FORTRAN code was run on a Hewlett-Packard workstation. Case studies
were conducted to check the validity of the model. The model
successfully reproduced the analytical solutions to the simple
diffusion problem and to the specialized case of diffusion with fast
reversible binding to unsaturable binding sites.
 |
RESULTS |
Heparin Binding Slows bFGF Transport--
The ability of bFGF to
bind HS is likely to have a significant impact on its transport through
an HS-rich matrix (12, 13). To gain insight into the role of bFGF
heparin binding in controlling transport, we conducted transport
studies with the non-heparin binding (inactive) bFGF fraction that was
isolated from the flow-through during heparin-Sepharose affinity
chromatography. This subfraction of 125I-bFGF likely
represented protein that had lost its ability to bind heparin as a
result of the labeling process or from the freeze/thaw involved in
storage. Thus inactive 125I-bFGF represented a chemical
analog of bFGF that was the same size and charge yet was structurally
altered such that it did not bind heparin/HS. The transport
characteristics of active (heparin binding) 125I-bFGF and
inactive 125I-bFGF across Descemet's membrane were
evaluated (Fig. 2). Over the time course
of these studies native bFGF that retained its capacity to bind heparin
was unable to cross the membrane with appreciable flux. The flux of the
non-heparin binding form of bFGF, normalized for membrane thickness and
bFGF source concentration, was approximately 20-fold greater than the
normalized flux of native bFGF. The non-binding bFGF had reached steady
state flux by 10 min, however, the native bFGF had still not reached
steady state even after 24 h (1440 min). The diffusion of bFGF
that bound heparin was dramatically reduced (>125-fold) compared with
the non-binding bFGF. Using a steady state analysis (see Equation 8) an
effective diffusivity of 7.0 × 10
9
cm2/s was calculated for the non-binding bFGF (Table
I). Effective diffusion coefficients
could not be determined for the native bFGF since steady state flux was
not reached over the time course analyzed.

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Fig. 2.
DM diffusion of heparin-binding and
non-heparin binding bFGF. The time course for diffusion of native
bFGF ( ) and non-heparin binding bFGF ( ) across two separate
pieces of the same DM is presented. Initial bFGF source concentrations
were set at 0.5 nM. Multiple samples were taken and
subjected to trichloroacetic acid precipitation and each fraction
(pellet, wash, supernatant) was counted and corrected for
experimentally determined crossover and the averaged
125I-bFGF concentrations in the source and sink chambers
were calculated for each time point. At the end of the experiment, the
membranes were removed and their thickness measured. Data presented
represent the average sink concentration normalized to the membrane
thickness and source chamber concentration
(csink·lmem/csource)
in one representative experiment. Multiple (3-6) counts of source or
sink chamber contents at given time points produced errors that were
smaller than the data points presented. The averaged effective
diffusivity for non-heparin binding bFGF was calculated to be 7.0 × 10 9 cm2/s. It was not possible to
determine an effective diffusivity from the native bFGF transport data
since steady state flux was not reached over this time course.
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Table I
Effective diffusion coefficients calculated from diffusion experiments
A steady state analysis of the diffusion data presented in Figs. 2 and
4-6 was conducted as described under "Experimental Procedures."
Briefly, the slope from the linear portion of a plot of flux
versus time was determined by a least squares fitting
routine. The product of the partition coefficient and effective
diffusion coefficient was determined by multiplying the flux by the
thickness of that particular membrane and by dividing the result by the
measured solute concentration in the source chamber. The
Deff was calculated using the independently measured
value of K. Values for K · Deff and Deff were determined
from each analysis and the averages ± S.E. (or range for
n = 2) of all experiments conducted under the indicated
condition are presented.
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In the diffusion experiments, bFGF was detected in the sink chamber by
measuring the trichloroacetic acid precipitable radioactivity. Detailed
analysis of the sink contents were performed after some experiments to
ensure that the radioactive protein accurately reflected the presence
of intact bFGF. Western blot analysis using anti-bFGF antibodies
identified a protein band at 18 kDa, identical to that of the native
bFGF starting material. Furthermore, autoradiographic analysis revealed
that the band was the only protein containing 125I-bFGF
(Fig. 3). Thus, trichloroacetic acid
precipitable radioactivity in the sink chamber accurately reflected the
presence of 125I-bFGF.

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Fig. 3.
Western blot and autoradiography of bFGF in
the sink chamber. 125I-bFGF in the sink chamber was
identified by Western blot and autoradiography. Sink chamber samples
were taken after a 24-h transport study with an initial
125I-bFGF source concentration of 1 µM.
Samples were subjected to reducing SDS-polyacrylamide gel
electrophoresis (15% running gel) and transferred to Immobilon P
membranes. Blots were hybridized with a monoclonal antibody to bFGF and
a single band with a relative molecular mass of 18 kDa was revealed by
chemiluminescence (lane 1). The chemiluminescence was
exhausted by 2-day incubation at room temperature and autoradiography
was conducted to identify radioactive protein (lane 2).
Subsequent sectioning of the Western blot filter itself, followed by
determining the radioactivity in a -counter, showed that all of the
radioactivity on the filter was associated with the 18-kDa bFGF.
Similar results were observed in one additional experiment.
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To confirm that HS was involved in modulating bFGF movement through DM,
membranes were predigested with heparinase I (10 units/ml, 20 h,
37 °C). Heparinase I was used since this enzyme targets the highly
sulfated (heparin-like) sequences within heparan sulfate that have been
implicated in bFGF binding (27). Diffusion studies with these membranes
and 125I-bFGF resulted in a 30% increase in bFGF
normalized flux (data not shown). Although transport limitations,
enzyme stability issues, and product inhibition prevented the enzyme
treatment from digesting a significant percentage of DM HS, this study
indicated that the HS retards native bFGF transport. Thus, the
specificity of the enzyme treatment confirmed that HS is an important
modulator of bFGF transport, however, additional methods were needed to
completely remove the HS binding component from the transport process
in order to obtain accurate values for the effective diffusivity parameter.
Decoupling Diffusion from Binding--
Several complementary
approaches were employed to obtain a reliable value for the effective
diffusivity (Deff) and to test our assumptions
about the nature of bFGF interaction within and movement through the DM.
The diffusion of bFGF was compared with that of IL-1
. IL-1
is a
cytokine of 17 kDa, with a pI of 7.0. Although it contains little
overall sequence homology with bFGF, the tertiary structure of IL-1
is very similar to bFGF, making bFGF and IL-1
structural homologs
(28). Structures from both solution NMR and x-ray crystallography show
that the two molecules have very similar molecular volumes (29, 30)
with bFGF having a diameter of approximately 29 Å and IL-1
having a
diameter of 32 Å. Hence, we assumed that the two proteins have similar
aqueous diffusivities. Consequently, IL-1
was used as a bFGF
"analog" that cannot bind heparin. Indeed, IL-1
demonstrated
very similar transport properties to the non-heparin binding bFGF
(Figs. 2 and 4). Based on this data and
the partition coefficients for IL-1
, an effective diffusivity for
IL-1
of 6.6 × 10
9 cm2/s was
obtained.

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Fig. 4.
bFGF and IL-1 DM
diffusion. Representative data for bFGF ( ) and IL-1 ( )
diffusion through DM in 3.0 M NaCl/PBS-BSA buffer and for
IL-1 in PBS-BSA at physiological ionic strength (×) are presented
(initial concentrations, 0.5 nM). Averaged effective
diffusivities for bFGF (3.8 × 10 9
cm2/s) and IL-1 (3.7 × 10 9
cm2/s) in 3.0 M NaCl and IL-1 (6.6 × 10 9 cm2/s) at physiological ionic strength
were calculated from multiple experiments. Fluxes in all experiments
were much higher than for heparin binding bFGF in PBS-BSA (Fig. 1). The
sink chamber concentration was determined as described in the legend to
Fig. 1 and under "Experimental Procedures," and has been normalized
to the individual membrane thicknesses and source chamber
concentrations
(csink·lmem/csource).
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If the association of native bFGF with heparan sulfate is disrupted,
the binding term is eliminated from the diffusion equation, and the
flux of native bFGF should be comparable to fluxes measured for
non-binding bFGF and IL-1
. High ionic strength disrupts general electrostatic interactions in the membrane, and since ionic
interactions are required for bFGF-HS binding, high salt concentrations
will prevent bFGF binding to HS (31, 32). The diffusion of bFGF and
IL-1
through Descemet's membrane was conducted in 3.0 M
NaCl PBS-BSA (Fig. 4). As expected, in a high salt environment, the flux of bFGF through the membrane was dramatically increased as compared with native bFGF in a physiological ionic strength buffer, by
preventing bFGF from associating with the HS in the DM. The normalized
flux of bFGF and IL-1
in 3.0 M NaCl were almost
identical. However, the normalized fluxes for both proteins in a 3.0 M NaCl/PBS-BSA buffer were approximately 60% of the
normalized flux of IL-1
in physiological saline buffer. In an
independent experiment, the DM was seen to reduce in thickness
approximately 10% in 3.0 M NaCl. The change in thickness
probably translated into a reduced effective pore size (16) and
resulted in reduced fluxes. The possibility that the high salt
extracted the HS chains from the matrix during the experiment was also
explored. Isolated Descemet's membranes were extracted overnight at
4 °C in 3.0 M NaCl and the supernatant was collected
after centrifugation. This supernatant was dialyzed against water with
3,000 MWCO tubing, lyophilized, and analyzed for heparan sulfate by
using selective lyase treatment and the dimethylmethylene blue dye
binding assay (33). No HS was present in the extract supernatant. Based
on the limits of detection of the heparan sulfate assay and the total
amount of HS present within the DM we calculate that
0.01% of DM HS
was extracted by 3.0 M NaCl.
Protamine sulfate binds heparan sulfate, thereby preventing the
association of bFGF with the HS chains (34). Consequently, protamine
sulfate was used to more specifically decouple bFGF binding from
diffusion without altering the bulk properties of the membrane.
Membranes were preincubated with protamine sulfate (10 mg/ml) for
48 h at 4 °C to ensure that all the HS sites in the membrane
were occupied, and the diffusion studies were subsequently conducted in
this concentration of protamine sulfate. With an average molecular mass
of approximately 6 kDa, the protamine sulfate concentration was 1.7 mM, over 1 million times the source concentration of bFGF.
The presence of protamine sulfate resulted in a dramatic increase in
bFGF flux (Fig. 5) as compared with the
flux of bFGF in PBS-BSA. The normalized flux of bFGF with protamine
sulfate was within 10% of the normalized flux of IL-1
. The
effective diffusivity of bFGF determined from this data was 5.8 × 10
9 cm2/s. The similar results for bFGF with
protamine sulfate and IL-1
diffusion were as expected. Both proteins
have similar molecular dimensions, and in the protamine sulfate
environment bFGF flux was not influenced by the HS in the DM.

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Fig. 5.
bFGF DM diffusion with protamine
sulfate. A representative set of data is presented for the
diffusion of bFGF (0.5 nM source concentration) through DM
in the presence of 10 mg/ml protamine sulfate ( ) in PBS-BSA buffer.
An averaged effective diffusivity (Deff 5.8 × 10 9 cm2/s) was calculated from multiple
experiments. The sink chamber concentration was determined as described
in the legend to Fig. 1 and under "Experimental Procedures," and
has been normalized to the individual membrane thickness and source
chamber concentrations
(csink·lmem/csource).
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Taken together the above data strongly implicates the HS-bFGF
interaction as the primary determinant of flux through the membrane. Theoretically, if experiments with the low concentrations of bFGF used
in Fig. 1 (0.5 nM) could be carried over very long time
periods (days to weeks), bFGF would be expected to eventually saturate HS sites in the membrane and then the bFGF flux would be equal to the
flux measured when the HS-bFGF interaction was blocked. However,
control experiments revealed that denaturation of bFGF within the mixed
diffusion chamber apparatus becomes significant between 24 and 48 h, making such experiments unfeasible. The approach to steady state can
be greatly accelerated using higher concentrations of bFGF. Thus,
transport was evaluated using a 2000-fold higher concentration of bFGF
(1 µM based on enzyme-linked immunosorbent assay) (Fig.
6). After a lag period of 11 h
during which the HS sites were occupied, the normalized flux rose to
approximately 80% of that derived from previous experiments with bFGF
and protamine sulfate (Fig. 5).

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Fig. 6.
Diffusion of 125I-bFGF with
excess unlabeled bFGF. The diffusion of 125I-bFGF (0.5 nM) through the DM with a 2000-fold excess of unlabeled
bFGF (total [bFGF] = 1 µM) ( ) showed lag times of
approximately 11 h followed by a normalized steady state flux
approaching that measured under conditions where bFGF association with
HS was blocked. The resulting averaged effective diffusivity calculated
from the steady state portion of these curves was 5.3 × 10 9 cm2/s. The sink chamber concentration was
determined as described in the legend to Fig. 1 and under
"Experimental Procedures," and has been normalized to the
individual membrane thicknesses and source chamber concentrations
(csink·lmem/csource).
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Numerical Model of bFGF Transport--
In order to determine if
the experimental results could be explained by the diffusion/binding
model, key parameter values were measured experimentally and
incorporated into a numerical solution of the diffusion/binding
equations (Equations 2-4). The diffusion/binding mathematical model
requires information about the kinetics of the bFGF-HS interaction and
the concentration of HS-binding sites. Thus, HS was extracted from the
DM by
-elimination using alkaline borohydride treatment, and the HS
was subsequently purified by ion exchange chromatography and
chondroitinase ABC and keratinase III/endo-
-galactosidase treatment.
The number averaged molecular mass of the purified DM HS was determined
to be 42 kDa. Based on the Mr and the calculated
purification yield, the concentration of HS in DM was determined to be
13 µM (relative error estimates based on initial
extraction yields indicate a possible range of 6.5-26
µM). bFGF binding to purified DM HS was conducted using a
rapid gel filtration method to separate and quantitate the relative
amount of bFGF bound to DM HS. Using this method the
Kd of the interaction of bFGF with DM HS was determined to be 23.6 ± 2.2 nM with a stoichiometry
of 4 mol of bFGF bound per mole of HS
chains.2 The binding kinetics
are fast, but minimum values were assigned to the
kon and koff of 4.2 × 105/M
1 s
1 and
1 × 10
2/s, respectively. The Damkholer numbers
calculated from these kinetics and the measured effective diffusivities
show that binding is, indeed, much faster than diffusion
(Daoff
30; Daon
1 × 104). Consequently, a local equilibrium will
always exist between soluble and bound bFGF at any position within the
membrane. These parameters were entered into the mathematical
diffusion/binding model and simulated experiments were conducted. The
parameter values, obtained from independent experiments, that were
included in the mathematical model are summarized in Table
II.
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Table II
Parameters used in numerical model of bFGF transport through DM
All the parameters used in this simulation were measured in independent
experiments (see Footnote 2). kon and
koff were related through the equilibrium binding
constant (Kd = 23.6 ± 2.2 nM).
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Fig. 7 shows the results of simulated
bFGF (1 µM, as in Fig. 6) diffusion experiments through
Descemet's membrane using the parameter values described above. These
simulations were conducted at a range of DM HS (6.5-26
µM) concentrations that included the measured value and
those at the extremes of the estimated error. For the concentration of
HS that we measured in the DM (13 µM), the model
predicted a lag time of ~11 h, followed by a constant steady state
flux that was within 10% of the experimental flux. Over the range of
HS concentrations analyzed, the steady state flux varied only slightly
while the predicted lag time ranged from 6.5 h at 6.5 µM, to 23 h at 26 µM HS. Thus within
the estimated errors of the various model parameters, the simulations
predicted transport data that were remarkably similar to the
experimental values (Figs. 6 and 7). These results suggest that the
transport of bFGF across a complex tissue can be represented with a
comparatively simple diffusion/binding model.

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Fig. 7.
Simulation of bFGF transport through DM.
Simulation of bFGF (1 µM source concentration) transport
through DM by numerically solving the diffusion/binding problem with
independent experimentally determined parameters (Table II) and a range
of DM HS concentrations presented. Simulations with the following
concentrations of HS are shown: 6.5 µM (dark thick
line), 13 (thick line), 19.5 µM
(thin line), and 26 µM (dashed
line). Simulations were performed on a Hewlett-Packard workstation
using the FORTRAN code described under "Experimental Procedures."
Predicted lag times for the range of HS concentrations tested were:
6.5, 11.5, 17.0, and 23 h, for the 6.5, 13, 19.5, and 26 µM conditions, respectively.
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DISCUSSION |
The generation of growth factor, hormone, and morphogen gradients
as a means to regulate cell function and differentiated state has
become increasing appreciated as an important regulatory mechanism
throughout biology (35-41). However, there remains little understanding of how such gradients are formed or stabilized over short
distances within developing and adult organisms. A general hypothesis
has been presented that the extracellular matrix might participate in
generating morphogen gradients (36, 38, 42, 43). In particular, the
direct binding of growth factors, such as members of the FGF and
transforming growth factor
families, with extracellular matrix
components has been suggested to be a critical element in cell and
tissue regulation (43). In the present study we have addressed the role
of the extracellular matrix as a regulator of growth factor transport
at a fundamental level. By focusing on a model system of bFGF transport
through an acellular extracellular matrix, Descemet's membrane, we
have identified diffusion with rapid reversible binding to resident heparan sulfate as the critical elements that control bFGF movement. Furthermore, we have established and tested mathematical models of bFGF
movement that can be applied to more general circumstances that are not
amenable to controlled experimentation.
Although the transport of bFGF through the extracellular matrix is a
complex process, we applied a simple model of solute diffusion with
reversible binding to resident matrix sites to this process. We
conducted experiments using the DM as a model to allow quantitative
measurements of the effective diffusivity of bFGF under various
conditions in which binding of bFGF to HS chains in the matrix was
either blocked or saturated (Figs. 2 and 4-6). Similar effective
diffusivities were measured in all cases (Table I). The results from
these studies, as well as direct determination of membrane HS
concentration, bFGF binding kinetics, and partition coefficients were
incorporated into a numerical solution of the diffusion/binding
equation set (Equations 2-4). Simulated diffusion experiments were
conducted to determine if these processes alone could accurately
explain our data. The experimental results were represented very well
by the diffusion/binding model (Figs. 6 and 7). Thus, our results
suggest that reversible binding of bFGF to resident heparan sulfate can
provide a mechanism for slow release, relative to the non-binding case,
of active bFGF in vivo that does not require enzymatic
degradation of the ECM or its components. However, in our studies where
bFGF binding was prevented, we observed an enormous acceleration of
bFGF transport. Hence, our data predicts that acute degradation of
matrix resident heparan sulfate would dramatically enhance bFGF release
rates. We saw no experimental evidence to support models where the
ability of bFGF to bind HS can accelerate bFGF movement through the
matrix. A mechanism for accelerated bFGF movement by HS might require greater concentrations of HS than we observed in the DM or simply might
not exist in the extracellular matrix.
Other studies on bFGF transport through extracellular matrix have
previously identified the interaction with heparin as critical to this
process. In a study by Dabin and Courtois (12), transport of bFGF
across a reconstituted basement membrane gel, MatrigelTM,
could be enhanced by increasing the bFGF concentration or by adding
soluble heparin. These investigators concluded that binding to resident
heparan sulfate sites functioned to slow bFGF movement and that
saturating these sites with bFGF or blocking the heparin-binding site
on bFGF with heparin could decrease bFGF-gel interactions. While the
present study is consistent with their hypothesis, the nature of the
experimental system used by Dabin and Courtois and the fact that
several key parameters (i.e. membrane thickness, HS
concentration) were not determined, prevent quantitative analysis and
direct comparison to the present study. Furthermore, work by
Flaumenhaft et al. (13) also concluded that binding of bFGF to resident charged sites within an insoluble matrix would limit bFGF's diffusion radius. In this study, bFGF diffusion in agarose or
fibrin gels was enhanced in the presence of soluble protamine, heparin,
or suramin, suggesting that these agents were disrupting ionic
interactions of bFGF with sites in the gels. Together these studies
suggested a diffusion with binding model for bFGF movement through
gels. In the present study we have extended these observations by
treating this process quantitatively in order to develop a generalized
mechanistic model of this transport process.
The experimental approach we employed using isolated pieces of DM
should be generally applicable to studying the diffusion of other
proteins or drugs through the basement membrane. Since this is the
first report of an experimental system that allows quantitative
measurements of solute transport through intact, nonextracted or
homogenized, basement membrane tissue specimens, the literature
contains no previous direct measurements of diffusivities in
Descemet's membrane for comparison. The flux of inulin (5,200 g/mol)
through corneal stroma with and without Descemet's membrane was
measured (44), and a permeability for the DM of 1.7 × 10
6 cm/s for 70-µm thick membranes was reported. If we
use our measured partition coefficient of 1.5, this permeability
corresponds to a Deff of 8 × 10
9 cm2/s for inulin in DM. Given the smaller
size of the inulin molecule, this value is in good agreement with the
diffusivities measured here for bFGF and IL-1
. Thus, data generated
with our experimental system is likely to be a good indicator of
basement membrane transport properties within the context of an intact tissue.
Diffusivities for a globular protein of bFGF's dimensions in free
aqueous solution (Do) would be expected to be
approximately 1.5 × 10
6 cm2/s using the
Stokes-Einstein equation based on a bFGF radius of 14.5 Å. Hence the
ratio Deff/Do is 0.004. This
analysis indicates that the structure of the DM presents a very
significant transport barrier to small proteins even in the absence of
binding. It is likely that the combination of the viscous drag and
tortuosity provided by the tight meshwork of protein and hydrated
polysaccharide within the membrane results in significantly retarded
protein diffusion (45).
The diffusivities reported here (Table I) were all based on the steady
state method. However, a lag time method can also be used to determine
effective diffusivities under conditions where binding does not occur.
The lag time (tlag) is defined as the
x-intercept of the line describing steady state flux in a plot of total solute in the sink chamber as a function of time (Deff = l2/6
tlag). However, this approach relies heavily on
gaining accurate data points at the early times, where error is
maximal, and is much more sensitive to varying membrane thickness than
the steady state approach. For these reasons the steady state method
produced more accurate values of Deff and was
the method of analysis we used. Nevertheless, effective diffusivities
determined using the lag time method were calculated (2 × 10
9 to 9 × 10
9 cm2/s) and
found to be in good agreement with the values obtained from the steady
state approach.
When heparan sulfate binding was allowed with high concentrations of
bFGF, lag times of 10 and 14 h were observed experimentally which
compared very well with a model lag time of 11.5 h (Figs. 6 and
7). The largest sources of error in the parameter values for the
simulations were in the concentration of HS sites. The effect of this
error was most pronounced on the lag time. Simulated lag times ranged
from 6 to 22 h when maximum and minimum values for the HS
concentration were used. However, across the entire error range of HS
concentrations, the steady state flux was within 10% of the
experimentally measured flux. These results indicate that the
diffusion/binding model accounts very accurately for the movement of
bFGF through the DM, and suggest that this model can be a useful tool
for exploring the role of ECMs in controlling the bioavailability of
bFGF in various tissue environments once the necessary parameters
(Table II) are known.
The diffusion/binding model can provide insights into the role of
basement membranes in regulating bFGF activity in vivo. Most
basement membranes are much thinner than Descemet's membrane and are
typically 50-100 nm thick. On this length scale, diffusion and binding
events will occur on similar time scales. Consequently, a 100-nm thick
basement membrane would be expected to behave very differently than a
50-µm thick Descemet's membrane with regard to its ability to act as
a bFGF reservoir. In order to determine the implications of our
diffusion/binding model for bFGF storage and release from thin basement
membranes, several simulations were conducted employing the model
parameters determined for Descemet's membrane. However, in this case,
a 100-nm thick membrane was initially saturated with bFGF. In the
simulation, this membrane is exposed to infinite sinks on both faces,
and the loss of bFGF from the membrane was tracked over time (Fig.
8). The concentration of bFGF along the
centerline of the membrane is plotted as a function of time. After 10 min virtually all the bFGF has been lost from the membrane.

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Fig. 8.
Simulated bFGF loss from 100-nm thick
basement membrane. The diffusion/binding model was used to
simulate the loss of bFGF from a saturated membrane 100 nm thick
exposed to infinite sinks on both faces (thick line). The
fractional saturation of the HS sites along the membrane's center line
is plotted as a function of time.
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