(Received for publication, March 9, 1995; and in revised form, November 10, 1995)
From the
The growth promoting activity of the subendothelial
extracellular matrix (ECM) is attributed to sequestration of basic
fibroblast growth factor (bFGF) by heparan sulfate proteoglycans and
its regulated release by heparin-like molecules and heparan sulfate
(HS) degrading enzymes. HS is also involved in bFGF receptor binding
and activation. The present study focuses on the growth promoting
activity and bFGF binding capacity of sulfate-depleted ECM. Corneal
endothelial cells (EC) maintained in the presence of chlorate, an
inhibitor of phosphoadenosine phosphosulfate synthesis, produced ECM
containing 10-15% of the sulfate normally present in ECM.
Incorporation of sulfate into HS was reduced by more than 90%. Binding
of I-bFGF to sulfate-depleted ECM was reduced by
50-60% and only about 10% of the ECM-bound bFGF was accessible to
release by heparin. Incubation of
I-bFGF on top of native
ECM resulted in dimerization of the ECM-bound bFGF, but there was a
markedly reduced binding and dimerization of bFGF on sulfate-depleted
ECM. ECM produced in the presence of chlorate contained a nearly
10-fold less endogenous bFGF as compared to native ECM and exerted
little or no mitogenic activity toward vascular EC and 3T3 fibroblasts.
In other studies, we investigated the interaction between
chlorate-treated vascular EC and either native or sulfate-depleted ECM.
Exogenous heparin stimulated the proliferation of chlorate-treated EC
seeded on native ECM, suggesting its interaction with ECM-bound bFGF
and subsequent presentation to high affinity cell surface receptors. On
the other hand, heparin had no effect on chlorate-treated cells seeded
in contact with sulfate-depleted ECM or regular tissue culture plastic.
Altogether, the present experiments indicate that heparan sulfate
proteoglycans associated with the cell surface and ECM act in concert
to regulate the bioavailability and growth promoting activity of bFGF.
While HS in the subendothelial ECM functions primarily in sequestration
of bFGF in the vicinity of responsive cells, HS on cell surfaces is
playing a more active role in displacing the ECM-bound bFGF and its
subsequent presentation to high affinity signal transducing receptors.
Heparan sulfate (HS) ()is a most ubiquitous
glycosaminoglycan present on cell surfaces, in basement membranes and
extracellular matrices (Gallagher et al., 1986; Jackson et
al., 1991; Kjellen and Lindahl, 1991). Recent interest in heparan
sulfate proteoglycans (HSPG) stems from increasing awareness of the
functional implications of their interactions with growth factors,
matrix molecules, and cytoskeletal elements (Gitay-Goren et
al., 1992; Jackson et al., 1991; Ruoslahti and Yamaguchi,
1991; Vlodavsky et al., 1993; Yayon et al., 1991). The HS chains have
been implicated in a variety of physiological processes including the
regulation of glomerular basement membrane permeability to proteins,
assembly of basement membranes, regulation of nuclear metabolism, cell
attachment and spreading, recruitment of inflammatory cells
(chemokines), and the regulation of mammalian cell proliferation and
differentiation (Gallagher et al., 1986; Jackson et
al., 1991; Ruoslahti and Yamaguchi, 1991; Tanaka et al.,
1993). The sulfate residues, which may be present on four different
positions of the polysaccharide backbone, are of high interest, since
they have been shown to be major factors in the determination of
specificity in protein-polysaccharide interactions (Lindahl, 1989). Of
particular significance is the interaction between HS and basic
fibroblast growth factor (bFGF), involved in bFGF receptor binding and
signal transduction (Ornitz et al., 1992; Rapraeger et
al., 1991; Yayon et al., 1991). A unique, highly sulfated
bFGF-binding fragment of HS was isolated from cell surface HSPG of
fibroblasts (Turnbull et al., 1992). Sulfation in critical
positions along the polysaccharide chain, particularly
2-O-sulfation, seems necessary to generate a specific bFGF
binding motif that can support high affinity bFGF-receptor binding and
activation (Aviezer et al., 1994b; Habuchi et al.,
1992; Ishihara et al., 1993; Maccarana et al., 1993;
Turnbull et al., 1992).
Chlorate, an inhibitor of ATP sulfurylase and hence of the production of phosphoadenosine phosphosulfate, the active sulfate donor for sulfotransferases (Baeuerle and Huttner, 1986), has been shown to abolish sulfation on proteins and carbohydrate residues in intact cells without inhibiting cell growth or protein synthesis (Baeuerle and Huttner, 1986; Keller et al., 1989). Exposure to chlorate markedly reduced binding of bFGF to high affinity cell surface receptors and the ability of 3T3 fibroblasts to proliferate in response to bFGF (Guimond et al., 1993; Rapraeger et al., 1991).
Our studies on the control of cell proliferation by its local environment focus on the interaction of cells with the extracellular matrix (ECM) produced by cultured corneal endothelial cells (EC) (Gospodarowicz et al., 1980; Vlodavsky et al., 1980, 1993). This ECM closely resembles the subendothelium in vivo in its morphological appearance and molecular organization. It contains collagens (mostly types III and IV, with smaller amounts of types I and V), proteoglycans (mostly HS- and dermatan sulfate-proteoglycans, with smaller amounts of chondroitin sulfate proteoglycans), laminin, fibronectin, entactin, and elastin. EC and other cell types plated in contact with this ECM no longer require the addition of soluble bFGF in order to proliferate and express their differentiated functions (Gospodarowicz et al., 1980; Vlodavsky et al., 1980). In subsequent studies bFGF was identified as a complex with HSPG in the subendothelial ECM produced in vitro (Bashkin et al., 1989; Vlodavsky et al., 1987) and on cell surfaces and basement membranes of diverse tissues and blood vessels (Cardon-Cardo et al., 1990; Gonzalez et al., 1990). HS-bound bFGF is protected against heat inactivation and proteolytic degradation (Saksela et al., 1988) and can be released in an active form by heparin-like molecules and HS degrading enzymes (Bashkin et al., 1989; Ishai-Michaeli et al., 1990, 1992; Vlodavsky et al., 1991), or by proteases (Benezra et al., 1993; Saksela et al., 1988). On the basis of these results, the ECM is regarded as a storage depot for bFGF and possibly other heparin-binding growth factors and cytokines. These immobilized growth factors are held responsible for the growth promoting activity of the ECM. In the present study, corneal EC were cultured in the presence of chlorate to produce sulfate-depleted ECM. This ECM was analyzed for its ability to sequester and dimerize bFGF and its growth promoting activity toward vascular EC and 3T3 fibroblasts, in the absence and presence of exogenously added heparin. We have also analyzed the ability of chlorate-treated EC to respond to native and sulfate-depleted ECM.
For preparation of sulfate-labeled ECM, corneal EC were
plated into four-well plates and cultured as described above.
NaSO
(540-590 mCi/mmol) was
added (20 µCi/ml) 1 and 5 days after seeding, and the cultures were
incubated with the label without medium change. Ten to 12 days after
seeding, the cell monolayer was dissolved and the ECM exposed, as
described above. To determine the total amount of sulfate labeled
material, the ECM was digested with trypsin (25 µg/ml, 24 h, 37
°C) and the solubilized material counted in a
-counter.
Protein was determined in aliquots of the trypsinized material using
the Coomassie protein assay reagent (Pierce) according to the
manufacturer's instructions. To determine the amount of sulfate
labeled HS, the ECM was digested (48 h, 37 °C, pH 6.2) with a human
placental heparanase (endo-
-D-glucuronidase) purified and
characterized as described (Gilat et al., 1995). The estimated M
of the HS fragments was 5,000-7,000 as
compared to a M
of about 30,000 ascribed to intact
HS side chains released from ECM by treatment with alkaline borohydride
or papain (Vlodavsky et al., 1983). Sulfate-labeled low M
degradation products released into the
incubation medium were analyzed by gel filtration on Sepharose 6B, as
described (Ishai-Michaeli et al., 1990).
Figure 1:
Effect of chlorate on sulfate
incorporation into HS in ECM. Corneal EC were seeded at a confluent
density (2 10
cells/16-mm well) in the absence
(
) or presence of increasing concentrations (
, 1
mM;
, 10 mM;
, 20 mM;
, 30
mM;
, 60 mM) of chlorate. The cells were
maintained in Fisher's medium in the presence of
Na
SO
(20 µCi/well) added on
day 1 and 5. ECM was prepared on day 10, followed by incubation (24 h,
37 °C, pH 6.2) with 2 µg/ml of a purified preparation of human
placental heparanase. Sulfate-labeled HS degradation products released
into the incubation medium were analyzed by gel filtration on Sepharose
6B. Inset, total amount of labeled sulfate determined
following trypsin digestion of the ECM. Aliquots of the trypsinized
material were counted in a
-counter. Each data point (cpm/well) is
the mean ± S.D. of four wells.
Figure 2:
Release of I-bFGF from ECM
produced in the absence and presence of chlorate. ECM produced in the
absence and presence of increasing concentrations of chlorate (legend
to Fig. 1) was incubated (3 h, 24 °C) with
I-bFGF (1.5
10
cpm/0.25 ml/well),
washed free of unbound bFGF and incubated (1 h, 24 °C) with 10
µg/ml heparin to displace the HS-bound bFGF. Radioactive material
released into the incubation medium was counted in a
-counter.
Released radioactivity is expressed as percent of
I-bound
to native ECM (6.5
10
cpm/well) and to ECM produced
in the presence of each concentration of chlorate . Each data point is
the mean of four wells. ``Spontaneous'' release of
I-bFGF in the presence of incubation medium alone was
subtracted from the experimental values.
We have previously demonstrated that EC and other cell types plated
in contact with the subendothelial ECM no longer require the addition
of soluble bFGF in order to proliferate (Gospodarowicz et al.,
1980; Vlodavsky et al., 1987). This mitogenic effect was
attributed primarily to the presence of bFGF in ECM, although the mode
of bFGF deposition was not elucidated (Vlodavsky et al.,
1987). ECM produced in the absence and presence of chlorate was tested
for mitogenic activity toward vascular EC and 3T3 fibroblasts. For this
purpose, vascular EC were seeded at a low cell density (1,000
cells/16-mm well) on top of ECM produced in the absence and presence of
30 mM chlorate. Six days after seeding, the cultures were
exposed to [H]thymidine and the amount of
trichloroacetic acid-precipitable radioactivity was determined 3 h
afterwards (Fig. 3A). EC were also seeded at a clonal
cell density (300 cells/35-mm dish) on top of native and
sulfate-depleted ECM and cell colonies were stained 10 days after
seeding (Fig. 3, inset). As demonstrated in Fig. 3A, ECM produced in the presence of chlorate
exerted a greatly reduced mitogenic activity toward vascular EC seeded
at a low or clonal cell density. In other experiments (Fig. 3B), ECM produced in the presence of increasing
concentrations of chlorate was subjected to trypsin digestion and
aliquots of the solubilized material were added to confluent,
growth-arrested 3T3 fibroblasts (Fig. 3B) or to
sparsely seeded EC (1,000 cells/16-mm well) maintained in the presence
of 10% heat-inactivated calf serum (data not shown). A trypsin digest
of native ECM was highly mitogenic to growth arrested 3T3 fibroblasts (Fig. 3B), and this activity was inhibited by
neutralizing anti-bFGF antibodies (data not shown). In contrast, ECM
produced in the presence of 30 mM chlorate was devoid of
mitogenic activity toward 3T3 fibroblasts (Fig. 3B).
Likewise, chlorate markedly reduced (
60%) the growth promoting
activity of ECM extracts toward sparsely seeded EC (data not shown). As
demonstrated in Fig. 3A, EC plated on chlorate-treated
ECM responded to exogenously added bFGF in a manner similar to cells
plated on regular tissue culture plastic. These results indicate that
sulfation is critical for the growth promoting activity of the ECM.
Figure 3:
Effect of chlorate on the mitogenic
activity of ECM toward vascular endothelial cells and 3T3 fibroblasts. A, vascular EC. Vascular EC were seeded at a low (1,000
cells/well) or clonal (300 cells/dish, inset) cell density on
tissue culture plastic, or ECM produced in the absence (ECM)
or presence (ECM + chlorate) of 30 mM chlorate.
The cells were maintained in DMEM containing 10% heat-inactivated calf
serum and tested for (i) thymidine incorporation, in the absence (striped box) and presence (shaded box) of 5 ng/ml
bFGF; and (ii) colony formation, in the absence of exogenously added
bFGF (inset, 1, plastic; 2, native ECM; 3, sulfate-depleted ECM), as described under
``Experimental Procedures.'' B, 3T3 fibroblasts. ECM
produced in the absence and presence of increasing concentrations of
chlorate was digested with trypsin (0.1 µg/ml, 3 h, 37 °C) and
aliquots (25 µl) of the solubilized material were tested for
induction of [H]thymidine incorporation in growth
arrested 3T3 fibroblasts. The basal incorporation of
[
H]thymidine into resting 3T3 fibroblasts was
<1,000 cpm as compared to 82,000 cpm in the presence of 0.5 ng/ml
bFGF. Each data point (cpm/well) is the mean ± S.D. of three
wells.
In other experiments, ECM produced in the absence and presence of 30 mM chlorate was digested (3 h, 37 °C) with 0.1 µg/ml trypsin and the amount of bFGF in the solubilized material was determined by an immunoassay (Quantikine human bFGF, R& Systems, Minneapolis, MN). The amount of bFGF in sulfate-depleted ECM was about 10-fold lower than that determined in native ECM (i.e. 11 and 121 pg of bFGF/ECM-coated 16-mm culture well, respectively). Similar results were obtained when the ECM was digested with bacterial (Flavobacterium heparinum) heparinase I (IBEX Technologies, Montreal, Canada) rather than trypsin. The heparinase-treated ECM exerted little or no mitogenic activity on vascular EC.
Figure 4:
Effect of heparin on bFGF sequestration
occurring when the EC layer is solubilized and the ECM exposed.
Confluent cultures (10 days after seeding) of corneal EC maintained in
the absence () and presence (
) of 30 mM chlorate
were exposed (5 min, 24 °C) to PBS containing 0.5% Triton X-100, 20
mM NH
OH,
I-bFGF (2
10
cpm/0.25 ml/well) and increasing concentrations of heparin. The
newly denuded ECM was washed three times, and the ECM-bound
radioactivity solubilized (1 N NaOH, 3 h, 37 °C) and
counted in a
-counter. Each data point is the mean ± S.D.
of four wells.
We next analyzed the effect of heparin on the
growth promoting activity of ECM produced in the absence and presence
of chlorate. Heparin, present during the 5-min cell lysis period, had
little or no effect on the growth promoting activity of native ECM
toward vascular EC seeded on ECM at a low (Fig. 5) or clonal
(data not shown) cell densities. Surprisingly, the mitogenic activity
toward EC of ECM produced in the presence of chlorate was stimulated
(1.5-4-fold, in different experiments) when heparin was included
in the cell lysis solution. This stimulation was observed both when the
endothelial cells were seeded directly on the ECM (Fig. 5) and
when the ECM was first digested with trypsin and aliquots of the
solubilized material were tested for mitogenic activity on vascular EC
(data not shown). Measurements of I-heparin binding
revealed that under the experimental conditions applied in Fig. 5, <0.5% of the heparin was bound to the ECM and there
was no difference in heparin binding to ECM produced in the absence or
presence of chlorate (data not shown).
Figure 5:
Effect of heparin on the mitogenic
activity of ECM produced in the absence and presence of chlorate.
Confluent corneal EC maintained in the absence () and presence
(
) of 30 mM chlorate were solubilized (5 min, 24 °C)
in Triton/NH
OH in the absence or presence of increasing
concentrations of heparin. Vascular EC were then seeded at a low cell
density (1,000 cells/well) in contact with the ECM and tested for DNA
synthesis ([
H]thymidine incorporation) on day 6
after seeding, as described under ``Experimental
Procedures.'' Each data point (cpm/well) is the mean ± S.D.
of four wells.
Figure 6: Effect of heparin on the growth of chlorate-treated endothelial cells. Vascular EC were pretreated for 24 h with 30 mM chlorate, dissociated with STV, and seeded (2,000 cells/16-mm well) in the presence of 30 mM chlorate into regular tissue culture wells (open box), and wells coated with ECM produced in the presence (shaded box) or absence (striped box) of chlorate. Heparin (1 µg/ml) was added to some of the wells on day 1 and 3 and the cells counted in a Coulter counter on day 5 after seeding. Each data point (cells/well) is the mean ± S.D. of four wells.
Figure 7:
Dimerization of bFGF on native and
sulfate-depleted ECM. I-bFGF (25 ng/0.25 ml/16-mm well)
was incubated (1 h, 24 °C) with regular tissue culture plastic,
native ECM, or ECM produced in the presence of 30 mM chlorate.
Heparin (5 µg/ml) was added to some of the wells (panel A, lanes 2, 4, and 6). The incubation medium
was aspirated, the dishes washed three times with PBS and incubated (30
min, 24 °C) with 0.15 mM DSS. The cross-linking reaction
was quenched with ethanolamine-HCl, as described under
``Experimental Procedures.'' The incubation medium was
removed and the bound
I-bFGF solubilized (150 µl of
sample buffer) and detected by 15% SDS-PAGE and autoradiography. A, same volume (60 µl) of solubilized material loaded on
each lane. Lanes 1 and 2, plastic; lanes 3 and 4, native ECM; lanes 5 and 6,
sulfate-depleted ECM. B, same amount of radioactivity
(
11,000 cpm) applied onto each lane. Lane 1, plastic; lane 2, native ECM; lane 3, sulfate-depleted ECM. M, monomer; D, dimer. Molecular size markers are
given in kilodaltons.
Heparan sulfates are heterogeneous molecules that vary both
in their basic disaccharide subunits and in their degree and position
of sulfation (Gallagher et al., 1986; Jackson et al.,
1991; Kjellen and Lindahl, 1991). Both the level of sulfation and
position of sulfate groups are major determinants in the interaction
between bFGF and HS and the ability of heparin and HS to promote bFGF
receptor binding and mitogenic activity (Aviezer et al.,
1994b; Habuchi et al., 1992; Ishihara et al., 1993;
Maccarana et al., 1993; Ornitz et al., 1992; Turnbull et al., 1992). Using chlorate, an inhibitor of
phosphoadenosine phosphosulfate synthesis, we investigated the
involvement of sulfate groups in the growth promoting activity of the
subendothelial ECM. Sulfate-depleted ECM exhibited a greatly reduced
mitogenic activity toward vascular EC and 3T3 fibroblasts, as compared
to native ECM. Similar results were obtained, regardless of whether the
vascular EC were seeded on top of chlorate-treated ECM or whether the
sulfate-depleted ECM was first digested with trypsin and aliquots of
the solubilized ECM added to EC seeded on regular tissue culture
plastic. The lack or low mitogenic activity may be due to (i) reduced
amounts of bFGF in ECM produced in the presence of chlorate and (ii)
inability of this ECM to present the ECM-bound bFGF to its high
affinity cell surface receptors. Measurements of bFGF binding revealed
a 50-60% reduction in bFGF binding to ECM produced by
chlorate-treated corneal EC, as compared to untreated cells. The amount
of HS-bound, heparin/heparinase releasable I-bFGF was
reduced by 70-80% in chlorate-treated ECM, suggesting that
I-bFGF may bind also to sulfate-depleted
glycosaminoglycan side chains (Ornitz et al., 1995), ECM
components other than HS (i.e. fibronectin), and possibly
ECM-bound bFGF receptors (Hanneken et al., 1995).
Direct immunoquantitation of bFGF in solubilized ECM revealed about a 10-fold reduction in the amount of endogenous bFGF in ECM produced in the presence of chlorate as compared to native ECM. In this assay the ECM was first digested with trypsin to solubilize the matrix and hence the reduced amounts of bFGF determined in sulfate-depleted ECM may be attributed, in part, to tryptic degradation of bFGF that is no longer protected by properly sulfated HS (Saksela et al., 1988). It should be noted, however, that a similar decrease in bFGF content was obtained when the ECM was digested with bacterial heparinase, rather than with trypsin, resulting in solubilization of >90% of the ECM-resident bFGF. These results, together with the lack of or greatly reduced mitogenic activity exerted by intact, undegraded sulfate-depleted ECM, suggest that this ECM exhibit little or no growth promoting activity simply because its HS chains fail to sequester bFGF and hence can not function as a secured depot of this growth factor in the vicinity of cells. Measurements of the cellular content of bFGF revealed no difference between chlorate-treated and untreated EC, suggesting that chlorate did not affect the synthesis of bFGF. An inhibitory effect on bFGF deposition, possibly as a complex with cell-associated HS, cannot be excluded.
A major concern in the study
of the growth promoting activity of the subendothelial ECM is whether
the ECM-bound bFGF is deposited into the ECM by intact EC, prior to
denudation of the ECM, or sequestered by HS and other components of the
ECM when the bFGF containing EC are lysed and the ECM exposed. In the
present study, heparin was included in the cell lysis solution in order
to eliminate the latter possibility. Measurements of I-bFGF binding revealed that heparin (10 µg/ml),
present in the cell lysis solution, inhibited by about 90% the binding
of
I-bFGF to the newly exposed native ECM. However, there
was no effect to this heparin on the growth promoting activity of the
ECM, indicating that the mitogenic activity of native ECM is not due to
sequestration of bFGF occurring when the ECM-producing cells are lysed.
An unexpected result was obtained when the effect of heparin on the
mitogenic activity of sulfate-depleted ECM, was investigated. Unlike
the results with native ECM, heparin, present during the 5-min cell
lysis period, stimulated the growth promoting activity of
sulfate-depleted ECM. A possible explanation for this stimulation is
the ability of heparin to bind to bFGF in the ECM and function in the
displacement and presentation of ECM-bound bFGF to high affinity
receptor sites on the cell surface. Alternatively, heparin may bind to
intracellular bFGF and then to the ECM, increasing the concentration of
bFGF in the undersulfated matrix. ECM binding of
I-heparin was low and there was no difference between
native and sulfate-depleted ECM, but this may result from the
iodination procedure, which could significantly alter the ability of
heparin to bind to heparin-binding proteins (i.e. fibronectin,
vitronectin) in the ECM. Heparin was previously shown to restore the
mitogenic response of chlorate-treated 3T3 fibroblasts and endothelial
cells to bFGF (Guimond et al., 1993; Rapraeger et
al., 1991).
Our experiments with chlorate-treated vascular EC
plated on intact native ECM, demonstrated that heparin can restore the
ability of the cells to proliferate in response to bFGF residing in the
ECM. Previous studies revealed that the K value
for interaction of bFGF with the cell surface HS (2
10
M) is lower than for interaction with HS
in the ECM (
1
10
M) (Bashkin et al., 1989; Moscatelli, 1987; Roghani et al.,
1994), suggesting that ECM-bound bFGF interacts first with HS on the
cell surface and is then presented to high affinity cell surface
receptors. Because the cell surface HSPG, unlike that of the ECM, is
mobile in the plane of the membrane and can turn over more rapidly by
shedding and internalization, it may readily replenish its bFGF from
the ECM reservoir, which serves more as an efficient large capacity
bFGF storage depot in the vicinity of cells (Bernfield and Hooper,
1991). Both functions (i.e. sequestration and presentation of
bFGF to high affinity receptor sites) may not be fulfilled by
non-sulfated HS side chains present in the ECM and surface of
chlorate-treated EC. A difference between cell surface- and ECM-
derived species of HS in their ability to promote bFGF mitogenicity was
also demonstrated in our recent studies on the growth promoting
activity of HS degradation fragments released by bacterial heparinase
III from ECM and cell surfaces. Using HS-deficient lymphoid cells, we
have demonstrated a stimulated cell proliferation induced by bFGF in
the presence of HS degradation fragments released from cell surfaces,
but not from ECM. (
)Altogether, it appears that HS in ECM,
unlike on cell surfaces, may not function efficiently as an accessory
low affinity receptor capable of directly accelerating the arrival of
bFGF at its high affinity signaling receptor.
The essential involvement of sulfate groups in bFGF receptor binding and activation was previously demonstrated by applying undersulfated and oversulfated species of heparin. These studies utilized chlorate-treated cells and HS-deficient cell mutants (Guimond et al., 1993; Rapraeger et al., 1991; Yayon et al., 1991). Best results were achieved in the presence of oversulfated heparin fragments, regardless of whether the N-position was sulfated or acetylated (Aviezer et al., 1994b). In a recent study (Ornitz et al., 1995), a stimulatory effect was also induced by synthetic, nonsulfated heparan-derived di- and trisaccharides. Our studies with native and sulfate-depleted endothelial cells and ECM demonstrate that properly sulfated HSPG associated with the cell surface and ECM act in concert to regulate the bioavailability of active bFGF and possibly other effector molecules to their signal transducing receptors.
Perlecan,
the large basement membrane proteoglycan, was recently identified as a
major candidate for a bFGF low affinity accessory receptor and an
angiogenic modulator. Other HSPG (e.g. syndecan, fibroglycan,
and glypican) exhibited only a small activity (Aviezer et al.,
1994a). Undersulfated perlecan synthesized in the presence of chlorate
is likely to exhibit a much lower capacity to sequester bFGF and
subsequently activate the high affinity bFGF cell surface receptor
site. This may result in a marked inhibition of the ECM-induced EC
proliferation and indirect involvement in neovascularization. It was
also demonstrated that acidic FGF binding to its low affinity accessory
receptor caused oligomerization of the FGF molecules, thereby
indirectly cross-linking and activating the high affinity receptors,
resulting in transmembrane signaling and cell proliferation
(Spivak-Kroizman et al., 1994). A similar ligand
oligomerization was observed during incubation of acidic FGF with
bovine lens epithelial cells (Mascarelli et al., 1993). In the
present study, intact ECM was found to induce dimerization of I-bFGF to a much higher extent as compared to
sulfate-depleted ECM. The markedly reduced dimerization was attributed
primarily to the decrease in bFGF binding and sequestration by HSPG in
the sulfate-deficient ECM. Dimerization of bFGF observed on
sulfate-depleted ECM may be mediated by both sulfated and nonsulfated
HS derived saccharides remaining in this ECM. The latter possibility
was recently reported (Ornitz et al., 1995). The highly
reduced ability of sulfate-depleted ECM to sequester and dimerize bFGF
is in all likelihood responsible for the impaired mitogenic activity of
this ECM. Oligomerization of ECM-bound bFGF and possibly other
heparin-binding growth factors may contribute to the potent growth- and
differentiation-promoting activities of the ECM. This oligomerization
is induced by properly sulfated HSPG found in native, but not
sulfate-depleted ECM. Specific alterations in the level and pattern of
sulfation along the HS side chains may thus provide a means to modulate
the involvement of HS and ECM in the control of cell proliferation and
differentiation and in processes such as neovascularization and tissue
remodeling.