Heparin and heparan sulfate (HS) constitute a class of glycosaminoglycans (GAGs) which can modulate various cellular functions such as cell growth, differentiation, morphology, and migration, etc. (Kjellen and Lindahl, 1991; Lindahl et al., 1994). Various growth factors known as heparin-binding growth factors such as fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), and midkine, etc. bind to cell surface HS (Ishihara and Ono, 1998). The binding to HS is important for their storage, release and stabilization. Furthermore, HS was found to function as a coreceptor of FGF. Prior to binding to the receptor, FGF must bind to HS (Rapreager et al., 1991; Yayon et al., 1991). This binding leads to the dimerization of FGF, which is required for the receptor activation (Spivak-Kroizman et al., 1994).
Vascular endothelial growth factors (VEGFs) are specific mitogens for vascular endothelial cells in vitro and angiogenic factor in vivo (Ferrara, 1996). They induce the permeabilization of blood vessels and are therefore also known as vascular permeability factors (VPFs). The VEGF family comprises secreted homodimeric proteins encoded by a single gene (Tisher et al., 1991). Alternative splicings of VEGF pre-mRNA lead to at least four peptides of 121, 165, 189, and 206 amino acids (Houck et al., 1991). The 121 amino acid form (VEGF121) differs from the larger VEGF isoforms in that it is the only VEGF type that does not bind to heparin and HS (Cohen et al., 1995; Gitay-Goren et al., 1996). To date, the best characterized VEGF isoform is the 165 amino acid form of vascular endothelial growth factor (VEGF165). In vascular endothelial cells, heparin or HS is required for efficient binding of VEGF165 to the VEGF receptors (Soker et al., 1994; Tessler et al., 1994).
The heparinoids (heparin, HS, modified heparin, and other heparin-like molecules) have microheterogeneities, and the structures may vary depending upon the cell type expressing them (Salmivirta et al., 1996). Their structures are made up of repeating units of disaccharides containing a uronic acid (UA) residue (either d-glucuronic acid or l-iduronic acid) and a glucosamine residue, which is either N-sulfated (GlcNS) or N-acetylated (GlcNAc). The disaccharides may be further sulfated at C6 and/or C3 of the d-glucosamine residue and C2 of the UA residue (Salmivirta et al., 1996). Using various chemically modified heparins, structural requirements in heparinoid for binding to FGFs have been elucidated. For example, FGF-2 is known to require N- and 2-O-sulfate groups in heparin for the binding, but does not require 6-O-sulfate groups (Habuchi et al., 1992; Turnbull et al., 1992; Ishihara, 1994b). In contrast with FGF-2, a high content of all three sulfate groups is required for its interaction with FGF-1 (Guimond et al., 1993; Ishihara, 1994b) or HGF (Lyon et al., 1994; Ashikari et al., 1995). However, nothing is known on the structural requirements in heparinoid for the interaction with VEGF165. In the present study, various chemically modified heparins were evaluated for their abilities to interact with VEGF165 using an ELISA procedure and to modulate the mitogenic activity of VEGF165.
LISA to evaluate the binding of glycosaminoglycan to heparin-binding growth factors
ELISA using heparin-immobilized beads was utilized in this work for evaluation of the interaction of chemically modified heparins with VEGF165. This assay measures an ability of heparin-derivatives to inhibit the binding of heparin-binding growth factors to heparin-beads. The bound growth factor to the heparin-beads is quantified by general ELISA procedure using a polyclonal antibody for the growth factor.
To show that FGF-2 (fibroblast growth factor-2), HGF (hepatocyte growth factor), and VEGF165 did bind to the heparin-beads and to determine a good working concentration for each growth factor, various concentrations of the growth factors (8 ng/100 µl to 0.1 ng/100 µl) were tested using this ELISA procedure (data not shown). Each growth factor bound to the beads in a concentration-dependent manner with maximum signal (OD [le] 0) at 8 ng/100 µl. The determined concentration for each growth factor (4 ng/100 µl) produced OD = 0.8 ~ 0.5. VEGF121, which is known not to bind to heparin, produced OD [le] 0.1 at the concentrations up to 8 ng/100 µl. Control wells in which the growth factor was not added also produced OD [le] 0.1. The mean OD from these triplicate determinations was used as the blank reference for the assays.
Ability of various glycosaminoglycans to inhibit the binding of each growth factor to heparin-beads
In this ELISA procedure, heparin or various polysaccharides that specifically bind to the growth factors would competitively inhibit the binding of the growth factor to the heparin-beads. As shown in Figure
Figure 1. Inhibitory effect of various glycosaminoglycans and modified heparins on the binding of FGF-2 to heparin-beads. (A) Chondroitin sulfate-A (CS-A), dermatan sulfate (DS), chondroitin sulfate-C (CS-C), and hyaluronic acid (HA) (all at 400 µg/ml) were tested for their ability to inhibit the binding of FGF-2 to the beads as described under Materials and methods. OD value on the binding of FGF-2 to the beads in the absence of any glycosaminoglycan was defined as 100% binding. The results represent the mean ± SE of triplicate determinations. (B) Various concentrations of heparin (solid diamonds), 2-O-DS-heparin (solid squares), 6-O-DS-heparin (solid triangles), N-DS,N-Ac-heparin (×) were tested for their ability to inhibit the binding of FGF-2 to the beads as described under Materials and methods. Binding of FGF-2 to the beads in the absence of any modified heparin was defined as 100% binding. The results represent the means ± SE of triplicate determinations.
Figure 2. Inhibitory effect of various glycosaminoglycans and modified heparins on the binding of HGF to heparin-beads. Various glycosaminoglycans (A) and modified heparins (B) were tested for their ability to inhibit the binding of HGF to the beads as described in the Figure 1 caption. Each symbol (B) represents each modified heparin as defined in the Figure 1 caption.
Figure 3. Inhibitory effect of various glycosaminoglycans and modified heparins on the binding of VEGF165 to the heparin-beads. Various glycosaminoglycans (A) and modified heparins (B) were tested for their ability to inhibit the binding of VEGF165 to the beads as described in the Figure 1 caption. Each symbol represents each modified heparin as defined in the Figure 1 caption. Effect of structural modifications of heparin
In order to study the structural features in heparin to specifically interact with FGF-2, HGF, and VEGF165, various chemically modified heparins were examined for their abilities to inhibit the bindings of those growth factors to the heparin-beads. Intact heparin had the ability to inhibit the bindings of all of FGF-2, HGF, and VEGF165 to the heparin-beads with IC50 values of about 25, 100, and 50 µg/ml, respectively (Figures
Table I.
Modification of O-sulfate groups on heparin resulted in changes in the inhibitory activities, and those changes were dependent upon the position and degree of desulfation. About 70% removal of 2-O-sulfate groups from the heparin (Table I) significantly reduced the ability to inhibit the bindings of FGF-2 and HGF to the beads (Figures Effect of chemically modified heparins to modulate VEGF165-induced cell growth
The chemically modified heparins were tested for their abilities to inhibit VEGF165-induced HUVEC growth (Figure
Figure 4. Inhibition of VEGF165-induced HUVEC growth by various modified heparins. HUVECs were grown for 3 days in medium 199 supplemented with 10% heat inactivated FBS, 4 ng/ml VEGF165, and various modified heparins at indicated concentrations. The cell growth was assessed by measurement of OD at 450 nm using WST-1 reagent as described under Materials and methods. The cell growth incubated with 4 ng/ml VEGF165 in the absence of any modified heparin was defined as 100% growth, and the data were calculated as percent growth. The broken line in the panel represents the level of cell growth obtained in the absence of both modified heparin and VEGF165. The results represent the means ± SE of triplicate determinations. Each symbol represents each modified heparin as defined in the Figure 1 caption.
Figure 5. Inhibition of VEGF165-induced HUVEC growth by high concentrations of modified heparins. HUVECs were grown for 3 days in medium 199 supplemented with 10% heat inactivated FBS, 4 ng/ml VEGF165, and various modified heparins at the indicated concentrations. The cell growth was assessed by measurement of OD at 450 nm using WST-1 reagent as described under Materials and methods. The cell growth incubated with 4 ng/ml VEGF165 in the absence of any modified heparin was defined as 100% growth, and the data were calculated as percent growth. The results represent the means ± SE of triplicate determinations.
Figure 6. Inhibition of FGF-2-induced HUVEC growth by high concentrations of modified heparins. HUVECs were grown for 3 days in medium 199 supplemented with 10% heat inactivated FBS, 2 ng/ml FGF-2, and various modified heparins at the indicated concentrations. The cell growth was assessed by measurement of OD at 450 nm using WST-1 reagent as described under Materials and methods. The cell growth incubated with 2 ng/ml FGF-2 in the absence of any modified heparin was defined 100% growth, and the data were calculated as percent growth. The results represent the means ± SE of triplicate determinations.
The high concentrations of 2-O-DS-heparin as well as native heparin were required to inhibit the VEGF165-induced cell growth, which might have caused some cell damage. In order to examine the possibility, HUVECs were grown in the presence of FGF-2 (2 ng/ml) and the high concentrations of native heparin, 2-O-DS-heparin, or 6-O-DS-heparin (256 and 512 µg/ml). As shown in Figure Effect of chemically modified heparins to restore VEGF165-induced proliferation of chlorate-treated HUVECs
Sodium chlorate, a potent inhibitor of sulfate adenyltransferase, reduces sulfations of various carbohydrates such as glycosaminoglycans (Humphries et al., 1988). It has been reported that chlorate blocks the abilities of FGF-1 and FGF-2 to stimulate the growth of endothelial cells (Ishihara et al., 1993) and fibroblast cells (Rapraeger et al., 1991), and that the chlorate blockage of FGF-mediated cell growth can be overcome by exogenous heparin. To see whether HUVECs respond similarly, the effect of chlorate on VEGF165-induced HUVEC growth was evaluated. When HUVECs were grown in medium lacking sulfate, the cells still responded to VEGF165 (Figure
Figure 7. Effect of chlorate on VEGF165-induced HUVEC growth. HUVECs were grown in DMEM lacking sulfate, but supplemented with 10% dialyzed FBS, 5 ng/ml VEGF165, 200 U/ml penicillin G, and other additions during indicated time periods. The cell growth was assessed by measurement of OD at 450 nm using WST-1 reagent as described under Materials and methods. The results represent the means ± SE of triplicate determinations. The other additions include: 15 mM sodium chlorate (solid squares), 15 mM sodium chlorate and 15 mM sodium sulfate (solid triangles), 15 mM sodium chlorate and 5 µg/ml heparin (×), 15 mM sodium chlorate, 15 mM sodium sulfate, and 5 µg/ml heparin (open circles), and no other addition (solid diamonds).
We have designed a mitogenic assay for an evaluation of various chemically modified heparins to restore the mitogenic activity of VEGF165 in chlorate-treated HUVECs as does heparin. HUVECs incubated in sulfate-free medium containing 5 ng/ml VEGF165 and 15 mM sodium chlorate did not grow (Figure
Figure 8. Restoration of VEGF-induced proliferation of chlorate-treated HUVECs by various modified heparins. HUVECs were grown in DMEM lacking sulfate, but supplemented with 10% dialyzed FBS, 15 mM sodium chlorate, 5 ng/ml VEGF165, 200 U/ml penicillin G, and various modified heparins at the indicated concentrations. The cell growth was assessed by measurement of OD at 450 nm using WST-1 reagent as described under Materials and methods. The cell growth in the absence of any modified heparin was defined as 100% growth, and each data were calculated as percent growth. The results represent the means ± SE of triplicate determinations. Each symbol represents each modified heparin as defined in the Figure 1 caption.
We previously utilized 2-O-DS- and 6-O-DS-heparins to examine the importance of 2-O-sulfate and 6-O-sulfate groups in promoting mitogenic activities mediated by FGF-1 and FGF-2 (Ishihara et al., 1995, 1997). Those studies showed that a high content of 6-O-sulfate groups is required for activation of FGF-1, but not FGF-2. The purpose of the present study was to examine the importance of each sulfate group in heparin for specific binding to VEGF165 and for modulation of its mitogenic activity, using the various chemically modified heparins. The major conclusions from this study are that a high content of 2-O-sulfate groups is not required for the specific binding to VEGF165, but that it is essential for its mitogenic activity.
The bindings of VEGF165, FGF-2, and HGF to the heparin-beads were strongly inhibited by native heparin, but chondroitin sulfate, dermatan sulfate, and hyaluronic acid did not inhibit the bindings in the ELISA assay. All of N-sulfate, 2-O-sulfate, and 6-O-sulfate groups in heparin were required for the binding to HGF (Asikari et al., 1995). However, it was described that 6-O-sulfate groups of heparin did not influence the interaction with FGF-2 (Guimond et al., 1993; Ishihara et al., 1994a). Thus, structural requirements for heparinoid to bind to FGF-2 are different from those to bind to HGF. In the present study, the removal of 6-O-sulfate groups from the heparin resulted in the significant loss of the abilities to inhibit the bindings of VEGF165 and HGF to the heparin-beads, but the 6-O-DS-heparin still retained the ability to inhibit the binding of FGF-2 to the heparin-beads. On the other hand, the removal of 2-O-sulfate groups from the heparin resulted in the significant loss of abilities to inhibit the bindings of FGF-2 and HGF to the heparin-beads, while the 2-O-DS-heparin still retained the ability to inhibit the binding of VEGF165 to the heparin-beads. Thus, these three growth factors appear to show intricate differences in their binding abilities on the selectively O-desulfated heparins. However, it is premature to conclude that 2-O-sulfate groups in heparin do not influence the interaction with VEGF165, since the 2-O-DS-heparin used in this work contained significant amount of trisulfated disaccharide units (14.4%) that might enhance the inhibitory ability.
The high concentrations of the native heparin (more than 64 µg/ml) could inhibit the VEGF165-induced HUVEC proliferations. Similarly, the 2-O-DS-heparin inhibited the cell proliferation (Figure
It has been reported that 2-O-DS-heparin as well as native heparin has heparanase-inhibitory, angiostatic, anti-tumor, and anti-metastatic activities (Lapierre et al., 1996). The formation of blood vessels is essential and also facilitates the dissemination of tumor cells to distant sites in the host. Heparanases can contribute to tumor angiogenesis by participating in hydrolysis of basement membrane and extracellular matrix (Nakajima et al., 1988). The report indicated that the mechanism of 2-O-DS-heparin to attenuate the new vessel formation did not include the interaction of 2-O-DS-heparin with an angiogenic factor, FGF-2. However, our presented results suggest that 2-O-DS-heparin can interact with another angiogenic factor (VEGF165) and can inhibit the activity of VEGF165, which may contribute to its anti-angiogenic activity.
It was shown that the interaction between VEGF165 and heparinoid is important for the interaction of VEGF165 with its receptors (Gitay-Goren et al., 1992; Soker et al., 1994; Tessler et al., 1994). Heparin can stimulate the binding of VEGF165 to its cell surface receptors in several cells. When the concentration of heparinoids on the cell surface is reduced following heparinase treatment, VEGF no longer binds to its cell surface receptors. The lack of binding is not due to a loss of VEGF receptors, since addition of exogenous heparin partially restores the binding capacity. VEGF121, a VEGF isoform lacking heparin-binding ability, also requires cell surface HS for its efficient binding to the VEGF receptors (Cohen et al., 1995). Thus, heparinoids play a critical role for biological activities of VEGFs.
In this study, we showed that VEGF165 significantly lost its mitogenic activity by the treatment of HUVECs with chlorate (Figure Materials
Human recombinant FGF-2, HGF, VEGF165, anti-FGF-2 (polyclonal, total rabbit IgG), anti-HGF (polyclonal, total goat IgG), and anti-VEGF (polyclonal, total goat IgG) were purchased from R&D Systems (Minneapolis, MN). Anti-goat (rabbit) IgG (horseradish peroxidase) and peroxidase substrate (ABTS) were purchased from Bio-Rad (Hercules, CA). Heparin-beads (type I) was purchased from Sigma (St. Louis, MO). Heparin, chondroitin sulfate-A, chondroitin sulfate-C, dermatan sulfate, and hyaluronic acid were obtained from Seikagaku Corp. Other chemicals were highest quality commercially available. Preparation of chemically modified heparins
N-Desulfated, N-acetylated heparin (N-DS, N-Ac-heparin):N-desulfated (N-DS) heparin was prepared according to the selective solvolytic N-desulfation method of Inoue and Nagasawa, 1976. The disaccharide compositional analysis treating this product with nitrous acid at pH 4 (Guo and Conrad, 1989) showed that neither O-desulfation nor other chemical changes had occurred during the reaction (data not shown). The prepared N-DS-heparin was then converted to the final product, N-DS, N-Ac-heparin, by the procedures described previously (Ishihara et al., 1997). Nitrous acid treatments at both pH 1.5 and 4 (Guo and Conrad, 1989) did not cleave the product at all (data not shown), indicating that the N-acetylation of N-DS-heparin was complete.
2-O-Desulfated (2-O-DS-) and 6-O-desulfated (6-O-DS-) heparins:2-O-DS-heparin (Ishihara et al., 1997) and 6-O-DS-heparin (Ishihara et al., 1995) were prepared by the methods reported previously. These procedures gave approximately 70% removal of 2-O-sulfates and 75% removal of 6-O-sulfates from the heparin, respectively (Table I). Gel-filtration chromatography showed no depolymerizations in the 2-O-DS- and 6-O-DS-heparins.
The compositional analyses of each modified heparin were performed as described previously (Yoshida et al., 1989; Kariya, et al., 1992). Briefly, 0.1 mg of a modified heparin was treated with a mixture of heparinase (50 mU), heparitinase I (20 mU), and heparitinase II (20 mU) (Seikagaku Corp.) in 220 µl of 2 mM calcium acetate and 20 mM sodium acetate (pH 7.0) at 37°C for 2 h. The disaccharide composition was determined by analyzing the reaction mixture by ion-exchange chromatography using Dinex CarboPacPA-1 (4 × 250 mm) and monitoring the optical absorbance at 230 nm for detection. The disaccharide compositions of each chemically modified heparins are shown in Table I. LISA for evaluating the ability of testing compounds to inhibit the bindings of the growth factors to the heparin-beads
In the 12-linked, 0.25 ml microtube (Nunc Inter Med, Tokyo, Japan), 100 µl of 1% BSA in PBS (BSA-PBS) including 4 ng of the human recombinant VEGF165, FGF-2, or HGF, and the indicated concentrations of testing polysaccharides were placed and mixed 30 min at room temperature. To each of the microtubes, 50 µl of the solution containing washed heparin-beads (1:1 mixture of heparin-agarose beads (Type I, Sigma) and polyacrylamide gel (Bio-Gel P-30, Bio-Rad)) was added and mixed for 30 min at room temperature. The heparin-beads in each tube were washed four times by centrifugation with the BSA-PBS containing the same concentration of the polysaccharides as before and by centrifugation four times with the PBS containing 0.02% Tween-20 (PBST). To the beads in each tube, 100 µl of anti-VEGF, anti-FGF-2, or anti-HGF, being diluted 1:500 with BSA-PBS, was added and mixed for 60 min at room temperature. The beads in each tube were washed four times by centrifugation with BSA-PBS and four times with PBST. To the beads in each tube, 100 µl of anti-IgG conjugates (horseradish peroxidase) diluted 1:1000 in BSA-PBS was added to the beads in each tube and mixed 60 min at room temperature. The beads were again washed four times by centrifugation with BSA-PBS and four times with PBST. Color was developed by adding 100 µl of solution containing horseradish peroxidase substrate (Bio-Rad) and by mixing for 30 min at room temperature. After centrifugation, the supernatant was transferred into each of 96 wells of the microtiter plate (Falcon) and the OD was read at 414 nm using the Immuno Mini Plate Reader (Nunc InterMed, Tokyo, Japan). Cell culture and proliferation assay
Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics, San Diego, CA. The HUVECs used in this work were between 4th to 6th passages. The cells were grown in medium 199 (Life Technologies, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 ng/ml FGF-2, and antibiotics (100 U/ml penicillin G and 100 µg/ml streptomycin) in atmosphere of 5% CO2 in air and 100% relative humidity, and were routinely subcultured at a split ratio of 1/5 or 1/6. For inhibition assay of VEGF165- or FGF-2-induced HUVEC growth, the cells were seeded at an initial density of 3000 cells/well in 96-well tissue culture plates and were grown for 3 days in 200 µl of medium 199 supplemented with 4 ng/ml VEGF165 or 2 ng/ml FGF-2, antibiotics, and various polysaccharides to be tested. For assay of chlorate-treated HUVEC, the cells were seeded at an initial density of 5000 cells/well and were grown for 3 days in DMEM lacking sulfate, but supplemented with 10% dialyzed FBS, 5 ng/ml VEGF165, 15 mM sodium chlorate, 200 U/ml penicillin G, and various polysaccharides to be tested. After the incubation the used medium was removed and 100 µl of the fresh medium including 10 µl of WST-1 reagent (Cell counting kit, Dojindo, Kumamoto, Japan) was added to each well, and the optical densities were read at 450 nm in Immuno Mini plate reader (Nunc InerMed Japan, Tokyo).
HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; HUVEC, human umbilical vein endothelial cell; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PBST, PBS with 0.02% Tween 20; 2-O-DS-, 2-O-desulfated; 6-O-DS-, 6-O-desulfated; N-DS, N-Ac-heparin, N-desulfated, N-acetylated heparin; OD, optical density; UA, uronic acid; GlcNS, N-sulfated glucosamine; GlcNAc, N-acetylated glucosamine; IC50, 50% inhibition concentration.
Heparin (%)
2-O-DS-heparin (%)
6-O-DS-heparin (%)
UA-GlcNAc
4.3
6.2
6.2
UA-GlcNS
3.8
20.5
5.3
UA-GlcAc(6-O-S)
0.7
2.9
0.3
UA(2-O-S)-GlcNAc
1.3
0
4.2
UA-GlcNS(6-O-S)
8.1
43.1
6.8
UA(2-O-S)-GlcNS
24.8
6.4
59.3
UA(2-O-S)-GlcNAc(6-O-S)
0
0
0.6
UA(2-O-S)-GlcNS(6-O-S)
52.6
14.4
12.7
Unknown
4.4
6.5
4.6
Discussion
Materials and methods
Abbreviations
References
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