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Binding of heparin to human microvascular endothelial cells and the effect on proliferation
Cell Biology
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Reports,
Vol. 12, No. 11, November
1988
931
BINDING OF HEPARIN TO HUMAN MICROVASCULAR ENDOTHELIAL CELLSAND THE EFFECT ON PROLIFERATION 1”
Andreas BIK ALVIl, EvelynelDUPUYl, Changgeng RUAN2, Gerard TOBELEM F Guy LESECHE , Jacques CAEN 1. INSERM U 150 and CNRS UA 334, Hijpital Lariboisiere, 75475 Paris Cedex 10, France 2. Thrombosis and Hemostasis Research Unit, Suzhou, China
ABSTRACT We have studied the binding of 125 I-heparin to human omental microvascular endothelial cells (HOME cells) and investigated its effect on cell proliferation. At ZO"C, the bin w reached a steady state from 4 hours onwards. Saturation of I-heparin binding occured at 200 nM. Scatchard anaixsis indicated one class of binding sites (KD = 0.023 NM, 2 X 10 Using fractionated sites/cell). heparin and other sulfated polysaccharides it was demonstrated that the binding was dependent on the charge and the molecular weight of the compounds. The binding was followed by partial the internalization of the bound ligand (23.8 X). Heparin and Stipocus Japonicus mucopolysaccharide (SJAMP) inhibited the proliferation of exponentially growing HOME cells and vein human umbilical endothelial cells (HUVE cells). However, serum-deprived HOME cells were not inhibited by heparin. INTRODUCTION Many interactions of heparin with vascular cells in culture have been demonstrated. Heparin is bound to human umbilical vein endothelial cells (HUVE cells) (Barzu et al., 1985, 1986), bovine capillary cells (Vannucchi et al., 1986) and smooth muscle cells et al., 1985) and is internalized. Heparin inhibits the (Castellot migration (Majak et Clowes, 1984) and the proliferation of smooth 1982). It stimulates the migration muscle cells (Castellot et al., of endothelial cells in the presence of endothelial cell growth 1985) and modulates their growth factor (ECGF) (Terranova et al., (Maciag et al., 1984 ; Schreiber et al., 1985 ; Rosenbaum et al., 1986). Heparin potentiates the mitogenic activity of heparin-binding growth factors-I (HBGF-I) (mainly ECGF and acidic fibroblast growth factor (aFGF)) by stabilizing their tertiary structure and enhancing their binding to the cell surface (Schreiber et al., 1985 ; Gospodarowicz et al., 1986). In et al., 1986b ; Gimenez-Gallego contrast, heparin inhibits the mitogenic activity of heparin binding growth factors-II (HBGF-II) (mainly basic fibroblast growth factor (bFGF)) and the binding (Gospodarowicz et al., 1986a ; Moscatelli, 1987). Capillary endothelial cells represent most of the vascular surface and play an important role in angiogenesis. However, the effect of heparin on these cells in culture has only little been
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investigated (Gospodarowicz et al., 1986a ; Vannuchi et al., 1986). In this report it is shown that heparin is bound with high affinity to human omental microvascular endothelial cells (HOME cells), that it is internalized and that it modulates proliferation. MATERIAL AND METHODS Reagents All chemicals for culture media and limbro 12 multiwell plates were purchased from Flow Laboratories (MC Lean, USA). Twenty four multiwell plates were obtained from Costar (Cambridge, USA). Ultroser G, a serum substitute, was from IBF (Villeuneuve la Garenne, France). Unfractionated standard heparin (standard porcine intestinal mucosal heparin, Mr 5,000 - 30,000 d) was obtained form the Choay Institute, (Paris, France). The low molecular fraction Fragmin (Mr range : 4,000 d to 6,000 d) was provided by Kabi (Kabi, Sweden). Stichopus japonicus acidic mucopolysaccharide (STAMP) (Mr range 40,000 d to 70,000 d) was kindly donated by Dr H.Z. Fan (Institute of materia medica, Tiensin, China). Molar concentrations were calculated using a mean Mr of 15,000 d for standard heparin, 5,000 d for fragmin and 55,000 d for SJAMP. Chondroitin sulfate (mixed isomers) (Mr 26,000), protease type XIV (pronase), triton X-100, sodium dodecylsulfate (SDS), and bovine serum albumin (BSA) were purchased from Sigma (St Louis, USA). Trypsin was obtained from OS1 (Paris, France). Collagenase was was purchased from Stago from Boehringer (Mannheim, FRG). Collagen Trichloroacetic acid (JCA), EDTA and EGTA were (Asnieres, France). H-methyl thymidine (20 France). obtained Prolabo (Paris, PiTrnI Na (13-17 Ci/mg iodide) were from Amersham (Les Ulis, Ci/mmol), France). Cell
cultures Human microvascular endothelial cells were isolated from the omental tissue (HOME cells) and cultured according to the technique Human umbilical vein endothelial described by Kern et al (1983). cells (HUVE cells) were obtained and cultured as previously 1973). Both cell types were grown in medium described (Jaffe et al., 199 (M 199) containing 20 % of fetal calf serum (FCS), 2 mm01 50 rig/ml streptomycin and 100 pg/ml glutamine, 50 U/ml penicillin, fungizone at 37°C in an humidified atmosphere with 5 % C02. The endothelial nature of both cells types was ascertained by a typical cobblestone appearance in confluent cultures and the presence of as demonstrated by factor VIII/van Willebrand factor antigen, indirect immunofluorescence. In HOME cells, only rare Weibel-Palade bodies and many microfilaments were demonstrated by electron microscopy according to the original description of Kern et al. where numerous Weibel-Palade in contrast to HUVE cells, (1983)s bodies were present. The cells were harvested with 0.025 % trypsin0.02 % EDTA in 20 mM phosphate buffered saline (PBS) pH 7.4 and subcultured at a split ratio of 1:3 in M 199 with 20 % FCS. For all experiments the cells were used between the first and third passage and grown on collagen coated plates.
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1988
Binding studies 125 Standard heparin was labeled with I Na according to the chloramine T method (Greenwood et al., 1963). The specific activity of labeled heparin was lo-15 mCi/mg. The binding experiments were performed as previously described (Barzu et al., 1985). The cells were grown to confluence in 24 multiwell plates in M 199 and 20 % FCS. Twenty four hours before the experiment the medium was removed, the cells were washed with serum free medium, and M 199 containing 2 % ultroser G was added. The binding experiments were performed in M 199 supplemented with 2 % ultroser G, which did not interfere with binding as shown earlier (Barzu et al., 1985). The cells were incubated with labeled heparin (l-5 pMiwel1 ; 200,000 - l,OOO,OOO cpmjwell) and non-labeled competitors for 4-5 h at 20°C. At the end of the incubation period, the cells were washed three times with PBS containing 0.1 % albumin and solubilized with pronase 0.1 % and triton X 100 0.1 % (v/v) solution. The radioactivity was counted For chasing experiments (Barzu et with a Beckman 7000 gammacounter. of labeled heparin was performed at 37°C and al., ; 985>, the binding after binding for 3 h with 4°C and the cells were incubated saturating concentrations of cold ligand (10 PM). After three washes with PBS containing 0.1 % albumin, the cells were solubilized and All experiments were done at least three the radioactivity counted. times in duplicates. Proliferation assays were seeded at 5 X LO4 Cell growth assay : HOME cells cells/well in 12 well plates in medium 199 with 20 % FCS. After were washed three times overnight attachment (< 24 h), the cells with serum free medium, and the test Jy%?g (various concentrations of FCS or 5 % FCS with or without 10 M-10 M heparin) was added to Control and test medium were replaced every two days. the wells. Duplicate wells were trypsinized after specific times intervals and the cells were counted with a coulter ZB-ZBI counter (Coultronics, The experiments were done three France) (2 determinations/well). times in duplicates and the results were expressed in percentages of proliferation according to the following formula : (cell number in heparin treated wells/cell number in control wells) X 100. Log-phase DNA synthesis assay : Confluent endothelial cells into 12 multiwell were passaged and plated at 5 X 104 cells/dish plates. After overnight attachment (< 24 h), the cells were washed intained with serum free mediurng2nldo_mgM heparin) in the test medium (5 % FCS 3 or with or without 10 H . Twenty four hours later, thymidine (3 uCi/well) were added for 24 h for HOME cells and 16 h for HUVE cells. The spent culture medium was then removed, and the cells were washed twice with 20 mM PBS pH 7.4 and precipitated twice with 10 % TCA. The TCA precipitable material was solubilized with a NaOH 0.25 M/SDS 0.1 % solution. The solution was transferred to and counted (Kontron Roche Bioelectronics, scintillation vials Trappes, France). Serum-deprived DNA synthesis assay : Cells were passaged and allowed to grow in M 199 with 20 % FCS for 3 to 4 days. Then the culture medium was removed and replaced by serum-deprived medium (M 199 with 0.5 % FCS) for 36 h. Subsequently the cells were washed
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1988
twice with-qeru_nb free medium and the test medium (5 % FCS with or without 10 -10 M of heparin) was added. Incorporation was achieved as in icated above. TJ H thymidine incorporation experiments were conducted three times in triplicates. The results were expressed in percentages relative to control calculated as follow : (cpm in heparin treated wells/cpm in control wells) X 100. RESULTS Binding of 125I-heparin to HOMEcell, The kinetics of binding of "'I-heparin at 20°C is shown in figure 1. Maximal binding was observed at 4 hours followed by a steady state. At equilibrium non-specific binding was usually very low and did not exceed 10 %.
1
2
3
4
5 time
(hrr)
Figure 1 : Kinetics of 1251-heparin binding to HOMECellT25Confluent I-labeled HOME cells were incubated at 20°C with 2 PM/well of At the indicated time intervals binding was standard heparin. in materials and methods, and the cellstopped as described associated radioactivity was determined (0-O ). Non-spef#z was determined by incubating cells with binding (+-+) heparin and a 500 fold excess of unlabeled ligand. The figure is representative of three experiments done in duplicates. binding was reached at 200 nM and the Saturation of 125I-heparin half maximum binding was 30 nM (fig.2). Analysis of the binding data according to Scatchard (1949) revealed one clags of binding sites with a dissociation constant of 23 nM and 2 X 10 binding sites/cell (fig. 3).
Cell Biology
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0.1 Total
Vol. 12, No. 11, November
0.3 Heparin(nM/weII)
1988
935
1
Figure 2 : Concentration dependance of 125 I-heparin binding to HOME cells. Monolayers of HOME cells were incubated with increasing concentrations of ligand at 2O'C. After 4 hours, the cells were cessed as indicated in material and methods and the cell-bound P?B I-heparin was determined. Non-specific binding was determined by a 500-fold excess of cold ligand. The figure is representative for three experiments done in duplicates,
B/F
0.01
‘251-Heparin (Pm/wall)
bound
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1988
125 Figure 3 : Scatchard plot of I-heparin binding data to HOME cells. Confluent HOME cells wps incubated as indicated in the legend of figure 2. Cell bound I-heparin was determined and the data were plotted according to Scatchard (1949). The figure is representative of three experiments done in duplicates. HOME YwI-heparin
cells were incubated with a fixed concentration of and increasing concentfg ions of unlabeled compound, the cold ligand ?Bpeted with I-heparin binding. At low concentrations of I-heparin (3 pM/ well) the half maximal displacement was of 0.03 JJM, which correlates reasonably well with the data obtained by Scatchard analysis. Unlabeled fractionated arin (fragmin) competed less than standard heparin with P% I-heparin binding (15U value of 0.5 PM) and no competition was with chondroitin sulfate (fig. 4). SJAMP competed strongly I-heparin binding to HOME cells (Is0 value of 0.02 DM).
100
8
-“~-~ 8
80 :
Glycosam;noglycan
( FM )
125 I-heparin binding to HOME cells by Competition of fragmin (M) ~~?~br~le!d gtandard heparin (H) SJAMP (w), sulfate (M). Confluent HOME 15711s were and chondroitin I-labeled incubated for 4 hours at 20°C with 2-5 PM/well of of increasing concentrations of standard heparin in the presence Results are expressed as the mean of a compounds. unlabeled duplicate experiment done four times. When saturating after binding dissociable at I>.
were added for 3 hours concentration o$ cold ligand was not at 37"C, 23.8 - 1.46 % of bound ligand 37'C suggesting a process of internalization (table
Cell Biology
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:;NBDLiEN:
;FEF;“l
Reports,
Vol. 12, No. 17, November
CT OF TEMPERATURE HEPARIN
Before (% of
ON
chasing control)
100
37oc 100
4OC
_’
TOTAL
AND
937
UNDISSOCIABLE
After (% of
chasing control)
23.8
+
-3.34
3.502
1988
1.46 ”
1.8
1251-heparinat
Confluent HOMEC were incubated for 4 h with 1 pM/well 4” or 37’C with or without further incubation for 3 h at 40 or 370~ with 10 PM unl+abeled heparin to measure the undissociable binding. results (mean - sem) are expressed Of three experiments in triplicates as percentages of the control binding at 37OC.
The
Effect
of serum and heparin on growth of HOME cells In the presence only of FCS (data not shown), the maximal with a was achieved the cell proliferation stimulation of concentration of 10 % serum. At maximal stimulation, the confluence was obtained in 8-10 days. Half maximal stimulation occurred at 3.5 % FCS. The doubling time in the presence of maximal stimulator-y concentrations of serum was 48-60 h. Heparin inhibited growth in HOME cells in the presence of 5 % PCS in a dose dependent manner as measured by cell counting at day eight (fig. 5). A proliferative rat_egof 47 % (53 % inhibition) was M. No-8inhibitcqy effect was observed at a concentration of 10 observed at lower heparin concentrations (10 M to 10 M).
25
Heparin
(M)
Figure 5 : Effect of standard heparin on the proliferation of HOME in the presence of 5 % FCS. The cells measured by cell counting,
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Vol. 12, No. 7 1, November
1988
experiments were performed as described in material and methods. The cell pumber was determined at day eight. The figure represents the mean - SEM of three experiments done in duplicates. Effect
of heparin on DNA synthesis of endothelial cells Log phase HOMEcells and HUVE cells : The effects of heparin and SJAMP-on the thymidine incorporation in log phase HOMEcells and HUVE cells in the presence of 5 % FCS wer-6 compared. The proliferative rate in the presence of heparin (10 M) was 65 % for HOMEcells (35 % of inhibition) and 58 % for HUVE cells (42 % of inhibition) (Table 2). The inhibitory effect was slightly higher for SJAMP in both cell types (Table 2). Serum deprived HOME cells : Heparin did not inhibit DNA synthesis in serum deprived HOME cells and even induced a slight stimulation (Table 3). TABLE
2
: INHIBITORY INDUCED BY
EFFECT 5 8 FCS
OF HEPARIN IN LOG PHASE
OR SJAMP ON ENDOTHELIAL
3H-THYhllDINE THE CELLS
HUVEC
HOhlEC Clycosaminoglycan Concentration
lo?
65.7
r
Heparin
SJAMP
Heparin
(Ml
3.77
INCORPORATION
SJAhlP
47.25
f
6.77
57.68
f
4.77
41.86
_+
5.57
66.29
i
8.96
86.119
f
8.96
+
0.19
lo-’
83.07
+
3.62
85.28
c
7.07
60.3
r
8.57
10 -8
104.8
?
3.65
-96.02
+
3.76
100.9
t_
7.1
1O-g
96.65
t
4.23
95.29
2
1.44
106.77
HOME cells or HUVE cells were seeded (5 X lo4 cells/well) in M 199 with 20_,% FCS-s After overnight - 10 hl of glycosaminoattachment, the test mediuys (M 199 containing 5 9, FCS with or without 10 H-thymidine incorporation was assessed as described in materials and glycans) wet-e added, and methods. The results (mean _’ sem) of three experiments done in triplicates were expressed as percentages of proliferation. TABLE 3 : EFFECT INCORPORATION HOME CELLS.
OF HEPARIN INDUCED BY
ON THE 3H-THYMIDINE 5 $ FCS IN SERUM DEPRIVED
Heparin CM)
Proliferation (%)
10 -6
115.21
:
10-7
129.65
_’ 6.97
1o-8
126.97
+ 14.2
10-9
119.13
:
3.02
10.6
HOME cells were seeded (5 X lo4 cells/well) in M 199 with 20 % FCS and allowed to grow for 3-4 days. The culture cells were maintained for 36 hours in serum deprived medium (M 199 with a 0.5 % FCSI. The test med_‘#m (M-d99 with 5 % FCSI in3the presence or absence of heparin - 10 Ml was added, an$l H-thymidine incorporation was (10 measured. The results (mean - sem) of three experiments done in triplicates were expressed as percentages of proliferation.
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1988
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DISCUSSION As previously shown in our laboratory for HUVE cells (Barzu et 1985) heparin bound to HOME cells in a saturable manner. When al, the binding data were analysed according to Scatchard (1949), it was shown that the dissociation constant was five times lower for HOME cells (KD of 0.023 yM) than the KD reported previously (Barzu et al, 1985) for high affinity binding sites of HUVE cells (KD of 0.12 PM). In contrast to HUVE cells, where two classes of heparin binding sites have been demonstrated, heparin bound to HOME cells via a single class of high affinity binding sites. The number of hepari -8 binding sites were found to be lower (3 X 10 for HOME cells sites/cell) than that reported for HUVE cells (1.16 X 10 binding sites/cell). Since heparin is heterogenous in its molecular weight (Mr of 5.000 - 30.000 d for the heparin used in the study) Scatchard analysis of the binding data gives only an Tygrage number of binding sites. Comparison of the binding data of I-heparin binding for HOME cells to that obtained by Castellot et al (1985) o-% smoott muscle cells (single class of binding sites, RD = 10 M, 10 sites/cell) indicates lower binding constants for hepari y2ginding to smooth muscle cells than to HOME cells. Thus, the I-heparin binding to HOME cells in terms of affinity and binding sites seems to be intermediate to that of smooth muscle cells and HUVE cells. Microvasculature, which represents 95 % of the total vascular surface, contains only 5 % of the entire blood volume which moves relatively slowly in this compartment. The high affinity of heparin for HOME cells may, therefore, have great implications for the pharmacokinetic of heparin in vivo. Much lower affinity was observed with low molecular weight heparin and no binding occurred with chondroitin sulfate, SJAMP which possess a high degree of sulfation et al., 1980) competed strongly and a high molecular weight (Fan with heparin binding to HOME cells. As demonstrated for HUVE cells (Barzu et al, 1985, 1986), the heparin binding for HOME cells seems to depend on the molecular weight and the degree of sulfation of the As found for other vascular cells (Barzu et glycosaminoglycans. 1985), there was only a partial al., 1985 ; Castellot et al., reversibility of the heparin binding at 37'C suggesting that a fraction of bound heparin (23.8 X) was internalized. Heparin seems to modulate the growth of endothelial cells. It inhibits the proliferation of HUVE cells at low concentration of 1986) and the proliferation of human serum (Rosenbaum et al., bovine capillary cells induced by HBGF-I and II (Gospodarowicz et al., 1986a). In contrast, it enhances mitogenesis in HUVE cells induced by HBGF-I (ECGF/aFGF) (Gospodarowicz et al., 1986a ; Maciag study, we et al., 1984 ; Schreiber et al., 1985). In the present have investigated heparin and SJAMR on the the activity of proliferation of HOME cells and compared the effect on DNA synthesis of exponentially growing HOME cells to that of HUVE cells. Heparin inhibited growth of HOME cells induced by 5 % FCS as measured by cell counting. In log-phase growing HOME cells and HUVE cells, heparin and SJAMP reduced DNA synthesis. The inhibitory effects of heparin and SJAMP on DNA synthesis was found to be of the same magnitude for HOME cells and for HUVE cells. This does not correlate with the differences in the affinities of both cell types for
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heparin. It has been previously shown for HUVE cells (Rosenbaum et weight heparin fragments retain a al, 1986) that low molecular similar inhibitory capacity than standard heparin in the presence of serum despite their lower binding affinities for HUVE cells. Thus, differences in the heparin binding affinities for endothelial cells may not imply differences in the inhibitory effects of heparin on endothelial cell proliferation. The reason for this is presently unknown. DNA synthesis of serum-deprived HOME cells was not inhibited by heparin. This may be in relation with different cellular densities in the log-phase and serum-deprived assay. Contradistinctive growth responses have also been observed for the effect of EGF on rat intestinal epithelial cells (Blay and Brown, 1987) and the effect of transforming growth factor on smooth muscle cells (Majak, 1987) where the proliferation of cells is inhibited in the exponential growth phase, but stimulated when cells reach high cellular densities. A conceptual framework regarding the effect of heparin on cell Several studies indicate that proliferation is not yet established. interfere with the binding of growth heparin binds to growth factors, and modify the internalization of the factors to their receptor, heparin biosynthesis. In endothelial cells, receptor or its stabilizes the tertiary structure of HBFG-I (ECGF/aFGF) (Schreiber 1986b) and increases its et al., et al., 1985 ; Gospodarowicz binding to the cell surface with exception of the brain derived growth factor for which the binding to bovine aortic endothelial cells is inhibited by heparin (Huang et al., 1985). In contrast, it inhibits the binding of HBGF-II (Moscatelli, 1987). In smooth muscle Reilly et al. (1987) have demonstrated that heparin inhibits cells, This the binding of EGF, but does not block the PDGF binding. inhibition is related to a decrease of EGF receptors induced by an intracellular action of heparin. In the present study, heparin exhibited an inhibitory effect on cell proliferation in the absence of HBGF-I and HBGF-II. The possibility that this effect is mediated by an intracellular mechanism as observed for smooth muscle cells is under investigation. Acknowledgements This work was supported by a grant from the "deutsche gemeinschaft" (No BI 332-l) for A.B.
Forschungs-
REFERENCES BARZU, T. , MOLHO, P., TOBELEM, G., PETITOU, M., and CAEN, J. (1985) Binding and endocytosis of heparin by human endothelial cells in culture. Biochem. Biophys. Acta, 845, 196-203 PETITOU, M., MOLHO, P., TOBELEM,G., BARZU, T., VAN RIJN, J.L.M.L., and CAEN, J. (1986) Endothelial binding sites for heparin. Biochem. J., 238, 1085-1097 BLAY, J. and BROWNK.D. (1986) Contradistinctive growth responses of cultured rat intestinal epithelial cells to epidermal growth factor depending on cell population density. J. Cell. Physiol., 129, 343-346
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CASTELLOT, J.J.L., FAVREAU, V., KARNOVSKY, M.J., and ROSENBERG, R.D. (1982) Inhibition of smooth muscle cell growth by endothelial cell-derived heparin : possible role of platelet endoglycosidase. J. Biol. Chem., 257, 11256-11260 CASTELLOT, J., WONG, K., HERMAN, B., HOOVER, R., ALBERTINI, D., WRIGHT, T., CALEB, B., and KARNOVSKY, M. (1985) Binding and internalization of heparin by vascular smooth muscle cells. J. Cell. Physiol., 124:13-20 FAN H.Z., CHEN J.D., and LIN, K.Z. (1980) An acidic mucropolysaccharide isolated from stipocus japonicus selenka and some of its physical and chemical properties. Yao Hseuh Hseuh Pao, 15, 263-270 GIMENEZ-GALLEGO, G., CONN, G., HATCHER, V.B., and THOMAS, K.A. (1986) Human brain derived acidic and basic fibroblast growth factors : amino terminal sequence and specific mitogenic activities. Biochem. Biophys. Res. Comm., 135, 541-54g5 GREENWOOD,F.C., HUNTER, W.H., and GLOVER, J.S. (1963) I-labeled growth hormone of high specific radioactivity. Biochem. J., 89, 114-123 GOSPODAROWICZ,D., MASSOGLIA, S., CHENG, J., and FUIJJI, D.K. (1986a) Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex an corpus luteum capillaries. J. Cell. Physiol., 127, 121-136 GOSPODAROWICZ,D. and CHENG, J. (1986b) Heparin protects basic and acidic FGF from inactivation. J. Cell. Physiol., 128, 475-484 HUANG, J.S., HUANG, S.S., and KUO, M.D. (1985) Bovine brain derived growth factor. Purification and characterization of its interaction with responsive cells. J. Biol. Chem., 260, 11600-11067 JAFFE, E.A., NACHMAN, R.L., BECKER, C.G., and MINICK, CV.R. (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest., 52, 2745-2756 and culture KERN, P., KNEDLER, A., and ECKEL, R.H. (1983) Isolation of microvascular endothelium from human adipose tissue. J. Clin. 71, 1822-1829 Invest., MACIAG, T., MEHLMAN, T. FRIESEL, R., and SCHREIBER, A.B. (1984) Heparin binds endothelial cell growth factor the principal endothelial mitogen in bovin brain. Science, 225, 932-935 MAJAK, R.A., and CLOWES, A.W. (1984) Inhibition of vascular smooth muscle cell migration by heparin-like glycosaminoglycans. J. Cell. Physiol., 118, 253-256 MOSCATELLI, D. (1987) High and low affinity binding sites for basic fibroblast growth factor on cultured cells : absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells. J. Cell. Physiol., 131, 131-140 ROSENBAUM, J., TOBELEM, G., MOLHO, P., BARZU, T., and CAEN, J. (1986) Modulation of endothelial cell growth by heparin. Cell Biol. Int. Rep., 10, 437-446
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7988
REILLY, C.F., FRITZE, L., and ROSENBERG,R.D. (1987) Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor. J. Cell. Physiol., 131, 149-157 SCATCHARD,G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci., 51, 660-672 SCHREIBER, A.B., KENNEY, J., KOWALSKI, W.J., FRIESEL, R., MEHLMAN, T.V. and MACIAG, T. (1985) Interaction of endothelial cell growth by receptor and antibody factor with heparin : characterization recognition. Proc. Natl. Acad. Sci. USA, 82, 6138-6142 TERRANOVA,V.P., DIFLORIA, R., LYALL, R.M., HIC, S., FRIESEL, R. and MACIAG, T. (1985) Human endothelial cells are chemotactic to endothelial cell growth factor and heparin. J. Cell. Biol., 101, 2330-2334 VANNUCHI, S., PASQUALI, F., CHIARUGI, V., RUGGIERO,M. (1986) Internalization and metabolism of endogenous heparin by cultured endothelial cells. Biochem. Biophys. Res. Comm., 140, 294-301
Received
26.8.88.
Accepted
29.9.88
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Vol. 12, No. 11, November
1988
931
BINDING OF HEPARIN TO HUMAN MICROVASCULAR ENDOTHELIAL CELLSAND THE EFFECT ON PROLIFERATION 1”
Andreas BIK ALVIl, EvelynelDUPUYl, Changgeng RUAN2, Gerard TOBELEM F Guy LESECHE , Jacques CAEN 1. INSERM U 150 and CNRS UA 334, Hijpital Lariboisiere, 75475 Paris Cedex 10, France 2. Thrombosis and Hemostasis Research Unit, Suzhou, China
ABSTRACT We have studied the binding of 125 I-heparin to human omental microvascular endothelial cells (HOME cells) and investigated its effect on cell proliferation. At ZO"C, the bin w reached a steady state from 4 hours onwards. Saturation of I-heparin binding occured at 200 nM. Scatchard anaixsis indicated one class of binding sites (KD = 0.023 NM, 2 X 10 Using fractionated sites/cell). heparin and other sulfated polysaccharides it was demonstrated that the binding was dependent on the charge and the molecular weight of the compounds. The binding was followed by partial the internalization of the bound ligand (23.8 X). Heparin and Stipocus Japonicus mucopolysaccharide (SJAMP) inhibited the proliferation of exponentially growing HOME cells and vein human umbilical endothelial cells (HUVE cells). However, serum-deprived HOME cells were not inhibited by heparin. INTRODUCTION Many interactions of heparin with vascular cells in culture have been demonstrated. Heparin is bound to human umbilical vein endothelial cells (HUVE cells) (Barzu et al., 1985, 1986), bovine capillary cells (Vannucchi et al., 1986) and smooth muscle cells et al., 1985) and is internalized. Heparin inhibits the (Castellot migration (Majak et Clowes, 1984) and the proliferation of smooth 1982). It stimulates the migration muscle cells (Castellot et al., of endothelial cells in the presence of endothelial cell growth 1985) and modulates their growth factor (ECGF) (Terranova et al., (Maciag et al., 1984 ; Schreiber et al., 1985 ; Rosenbaum et al., 1986). Heparin potentiates the mitogenic activity of heparin-binding growth factors-I (HBGF-I) (mainly ECGF and acidic fibroblast growth factor (aFGF)) by stabilizing their tertiary structure and enhancing their binding to the cell surface (Schreiber et al., 1985 ; Gospodarowicz et al., 1986). In et al., 1986b ; Gimenez-Gallego contrast, heparin inhibits the mitogenic activity of heparin binding growth factors-II (HBGF-II) (mainly basic fibroblast growth factor (bFGF)) and the binding (Gospodarowicz et al., 1986a ; Moscatelli, 1987). Capillary endothelial cells represent most of the vascular surface and play an important role in angiogenesis. However, the effect of heparin on these cells in culture has only little been
*
To whom correspondance
0309-1651/68/110931-12/$03.00/O
should
be addressed. @ 1988 Academic
Press Ltd.
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Vol. 72, No. 1 I, November
1988
investigated (Gospodarowicz et al., 1986a ; Vannuchi et al., 1986). In this report it is shown that heparin is bound with high affinity to human omental microvascular endothelial cells (HOME cells), that it is internalized and that it modulates proliferation. MATERIAL AND METHODS Reagents All chemicals for culture media and limbro 12 multiwell plates were purchased from Flow Laboratories (MC Lean, USA). Twenty four multiwell plates were obtained from Costar (Cambridge, USA). Ultroser G, a serum substitute, was from IBF (Villeuneuve la Garenne, France). Unfractionated standard heparin (standard porcine intestinal mucosal heparin, Mr 5,000 - 30,000 d) was obtained form the Choay Institute, (Paris, France). The low molecular fraction Fragmin (Mr range : 4,000 d to 6,000 d) was provided by Kabi (Kabi, Sweden). Stichopus japonicus acidic mucopolysaccharide (STAMP) (Mr range 40,000 d to 70,000 d) was kindly donated by Dr H.Z. Fan (Institute of materia medica, Tiensin, China). Molar concentrations were calculated using a mean Mr of 15,000 d for standard heparin, 5,000 d for fragmin and 55,000 d for SJAMP. Chondroitin sulfate (mixed isomers) (Mr 26,000), protease type XIV (pronase), triton X-100, sodium dodecylsulfate (SDS), and bovine serum albumin (BSA) were purchased from Sigma (St Louis, USA). Trypsin was obtained from OS1 (Paris, France). Collagenase was was purchased from Stago from Boehringer (Mannheim, FRG). Collagen Trichloroacetic acid (JCA), EDTA and EGTA were (Asnieres, France). H-methyl thymidine (20 France). obtained Prolabo (Paris, PiTrnI Na (13-17 Ci/mg iodide) were from Amersham (Les Ulis, Ci/mmol), France). Cell
cultures Human microvascular endothelial cells were isolated from the omental tissue (HOME cells) and cultured according to the technique Human umbilical vein endothelial described by Kern et al (1983). cells (HUVE cells) were obtained and cultured as previously 1973). Both cell types were grown in medium described (Jaffe et al., 199 (M 199) containing 20 % of fetal calf serum (FCS), 2 mm01 50 rig/ml streptomycin and 100 pg/ml glutamine, 50 U/ml penicillin, fungizone at 37°C in an humidified atmosphere with 5 % C02. The endothelial nature of both cells types was ascertained by a typical cobblestone appearance in confluent cultures and the presence of as demonstrated by factor VIII/van Willebrand factor antigen, indirect immunofluorescence. In HOME cells, only rare Weibel-Palade bodies and many microfilaments were demonstrated by electron microscopy according to the original description of Kern et al. where numerous Weibel-Palade in contrast to HUVE cells, (1983)s bodies were present. The cells were harvested with 0.025 % trypsin0.02 % EDTA in 20 mM phosphate buffered saline (PBS) pH 7.4 and subcultured at a split ratio of 1:3 in M 199 with 20 % FCS. For all experiments the cells were used between the first and third passage and grown on collagen coated plates.
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1988
Binding studies 125 Standard heparin was labeled with I Na according to the chloramine T method (Greenwood et al., 1963). The specific activity of labeled heparin was lo-15 mCi/mg. The binding experiments were performed as previously described (Barzu et al., 1985). The cells were grown to confluence in 24 multiwell plates in M 199 and 20 % FCS. Twenty four hours before the experiment the medium was removed, the cells were washed with serum free medium, and M 199 containing 2 % ultroser G was added. The binding experiments were performed in M 199 supplemented with 2 % ultroser G, which did not interfere with binding as shown earlier (Barzu et al., 1985). The cells were incubated with labeled heparin (l-5 pMiwel1 ; 200,000 - l,OOO,OOO cpmjwell) and non-labeled competitors for 4-5 h at 20°C. At the end of the incubation period, the cells were washed three times with PBS containing 0.1 % albumin and solubilized with pronase 0.1 % and triton X 100 0.1 % (v/v) solution. The radioactivity was counted For chasing experiments (Barzu et with a Beckman 7000 gammacounter. of labeled heparin was performed at 37°C and al., ; 985>, the binding after binding for 3 h with 4°C and the cells were incubated saturating concentrations of cold ligand (10 PM). After three washes with PBS containing 0.1 % albumin, the cells were solubilized and All experiments were done at least three the radioactivity counted. times in duplicates. Proliferation assays were seeded at 5 X LO4 Cell growth assay : HOME cells cells/well in 12 well plates in medium 199 with 20 % FCS. After were washed three times overnight attachment (< 24 h), the cells with serum free medium, and the test Jy%?g (various concentrations of FCS or 5 % FCS with or without 10 M-10 M heparin) was added to Control and test medium were replaced every two days. the wells. Duplicate wells were trypsinized after specific times intervals and the cells were counted with a coulter ZB-ZBI counter (Coultronics, The experiments were done three France) (2 determinations/well). times in duplicates and the results were expressed in percentages of proliferation according to the following formula : (cell number in heparin treated wells/cell number in control wells) X 100. Log-phase DNA synthesis assay : Confluent endothelial cells into 12 multiwell were passaged and plated at 5 X 104 cells/dish plates. After overnight attachment (< 24 h), the cells were washed intained with serum free mediurng2nldo_mgM heparin) in the test medium (5 % FCS 3 or with or without 10 H . Twenty four hours later, thymidine (3 uCi/well) were added for 24 h for HOME cells and 16 h for HUVE cells. The spent culture medium was then removed, and the cells were washed twice with 20 mM PBS pH 7.4 and precipitated twice with 10 % TCA. The TCA precipitable material was solubilized with a NaOH 0.25 M/SDS 0.1 % solution. The solution was transferred to and counted (Kontron Roche Bioelectronics, scintillation vials Trappes, France). Serum-deprived DNA synthesis assay : Cells were passaged and allowed to grow in M 199 with 20 % FCS for 3 to 4 days. Then the culture medium was removed and replaced by serum-deprived medium (M 199 with 0.5 % FCS) for 36 h. Subsequently the cells were washed
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1988
twice with-qeru_nb free medium and the test medium (5 % FCS with or without 10 -10 M of heparin) was added. Incorporation was achieved as in icated above. TJ H thymidine incorporation experiments were conducted three times in triplicates. The results were expressed in percentages relative to control calculated as follow : (cpm in heparin treated wells/cpm in control wells) X 100. RESULTS Binding of 125I-heparin to HOMEcell, The kinetics of binding of "'I-heparin at 20°C is shown in figure 1. Maximal binding was observed at 4 hours followed by a steady state. At equilibrium non-specific binding was usually very low and did not exceed 10 %.
1
2
3
4
5 time
(hrr)
Figure 1 : Kinetics of 1251-heparin binding to HOMECellT25Confluent I-labeled HOME cells were incubated at 20°C with 2 PM/well of At the indicated time intervals binding was standard heparin. in materials and methods, and the cellstopped as described associated radioactivity was determined (0-O ). Non-spef#z was determined by incubating cells with binding (+-+) heparin and a 500 fold excess of unlabeled ligand. The figure is representative of three experiments done in duplicates. binding was reached at 200 nM and the Saturation of 125I-heparin half maximum binding was 30 nM (fig.2). Analysis of the binding data according to Scatchard (1949) revealed one clags of binding sites with a dissociation constant of 23 nM and 2 X 10 binding sites/cell (fig. 3).
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0.1 Total
Vol. 12, No. 11, November
0.3 Heparin(nM/weII)
1988
935
1
Figure 2 : Concentration dependance of 125 I-heparin binding to HOME cells. Monolayers of HOME cells were incubated with increasing concentrations of ligand at 2O'C. After 4 hours, the cells were cessed as indicated in material and methods and the cell-bound P?B I-heparin was determined. Non-specific binding was determined by a 500-fold excess of cold ligand. The figure is representative for three experiments done in duplicates,
B/F
0.01
‘251-Heparin (Pm/wall)
bound
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1988
125 Figure 3 : Scatchard plot of I-heparin binding data to HOME cells. Confluent HOME cells wps incubated as indicated in the legend of figure 2. Cell bound I-heparin was determined and the data were plotted according to Scatchard (1949). The figure is representative of three experiments done in duplicates. HOME YwI-heparin
cells were incubated with a fixed concentration of and increasing concentfg ions of unlabeled compound, the cold ligand ?Bpeted with I-heparin binding. At low concentrations of I-heparin (3 pM/ well) the half maximal displacement was of 0.03 JJM, which correlates reasonably well with the data obtained by Scatchard analysis. Unlabeled fractionated arin (fragmin) competed less than standard heparin with P% I-heparin binding (15U value of 0.5 PM) and no competition was with chondroitin sulfate (fig. 4). SJAMP competed strongly I-heparin binding to HOME cells (Is0 value of 0.02 DM).
100
8
-“~-~ 8
80 :
Glycosam;noglycan
( FM )
125 I-heparin binding to HOME cells by Competition of fragmin (M) ~~?~br~le!d gtandard heparin (H) SJAMP (w), sulfate (M). Confluent HOME 15711s were and chondroitin I-labeled incubated for 4 hours at 20°C with 2-5 PM/well of of increasing concentrations of standard heparin in the presence Results are expressed as the mean of a compounds. unlabeled duplicate experiment done four times. When saturating after binding dissociable at I>.
were added for 3 hours concentration o$ cold ligand was not at 37"C, 23.8 - 1.46 % of bound ligand 37'C suggesting a process of internalization (table
Cell Biology
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:;NBDLiEN:
;FEF;“l
Reports,
Vol. 12, No. 17, November
CT OF TEMPERATURE HEPARIN
Before (% of
ON
chasing control)
100
37oc 100
4OC
_’
TOTAL
AND
937
UNDISSOCIABLE
After (% of
chasing control)
23.8
+
-3.34
3.502
1988
1.46 ”
1.8
1251-heparinat
Confluent HOMEC were incubated for 4 h with 1 pM/well 4” or 37’C with or without further incubation for 3 h at 40 or 370~ with 10 PM unl+abeled heparin to measure the undissociable binding. results (mean - sem) are expressed Of three experiments in triplicates as percentages of the control binding at 37OC.
The
Effect
of serum and heparin on growth of HOME cells In the presence only of FCS (data not shown), the maximal with a was achieved the cell proliferation stimulation of concentration of 10 % serum. At maximal stimulation, the confluence was obtained in 8-10 days. Half maximal stimulation occurred at 3.5 % FCS. The doubling time in the presence of maximal stimulator-y concentrations of serum was 48-60 h. Heparin inhibited growth in HOME cells in the presence of 5 % PCS in a dose dependent manner as measured by cell counting at day eight (fig. 5). A proliferative rat_egof 47 % (53 % inhibition) was M. No-8inhibitcqy effect was observed at a concentration of 10 observed at lower heparin concentrations (10 M to 10 M).
25
Heparin
(M)
Figure 5 : Effect of standard heparin on the proliferation of HOME in the presence of 5 % FCS. The cells measured by cell counting,
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1988
experiments were performed as described in material and methods. The cell pumber was determined at day eight. The figure represents the mean - SEM of three experiments done in duplicates. Effect
of heparin on DNA synthesis of endothelial cells Log phase HOMEcells and HUVE cells : The effects of heparin and SJAMP-on the thymidine incorporation in log phase HOMEcells and HUVE cells in the presence of 5 % FCS wer-6 compared. The proliferative rate in the presence of heparin (10 M) was 65 % for HOMEcells (35 % of inhibition) and 58 % for HUVE cells (42 % of inhibition) (Table 2). The inhibitory effect was slightly higher for SJAMP in both cell types (Table 2). Serum deprived HOME cells : Heparin did not inhibit DNA synthesis in serum deprived HOME cells and even induced a slight stimulation (Table 3). TABLE
2
: INHIBITORY INDUCED BY
EFFECT 5 8 FCS
OF HEPARIN IN LOG PHASE
OR SJAMP ON ENDOTHELIAL
3H-THYhllDINE THE CELLS
HUVEC
HOhlEC Clycosaminoglycan Concentration
lo?
65.7
r
Heparin
SJAMP
Heparin
(Ml
3.77
INCORPORATION
SJAhlP
47.25
f
6.77
57.68
f
4.77
41.86
_+
5.57
66.29
i
8.96
86.119
f
8.96
+
0.19
lo-’
83.07
+
3.62
85.28
c
7.07
60.3
r
8.57
10 -8
104.8
?
3.65
-96.02
+
3.76
100.9
t_
7.1
1O-g
96.65
t
4.23
95.29
2
1.44
106.77
HOME cells or HUVE cells were seeded (5 X lo4 cells/well) in M 199 with 20_,% FCS-s After overnight - 10 hl of glycosaminoattachment, the test mediuys (M 199 containing 5 9, FCS with or without 10 H-thymidine incorporation was assessed as described in materials and glycans) wet-e added, and methods. The results (mean _’ sem) of three experiments done in triplicates were expressed as percentages of proliferation. TABLE 3 : EFFECT INCORPORATION HOME CELLS.
OF HEPARIN INDUCED BY
ON THE 3H-THYMIDINE 5 $ FCS IN SERUM DEPRIVED
Heparin CM)
Proliferation (%)
10 -6
115.21
:
10-7
129.65
_’ 6.97
1o-8
126.97
+ 14.2
10-9
119.13
:
3.02
10.6
HOME cells were seeded (5 X lo4 cells/well) in M 199 with 20 % FCS and allowed to grow for 3-4 days. The culture cells were maintained for 36 hours in serum deprived medium (M 199 with a 0.5 % FCSI. The test med_‘#m (M-d99 with 5 % FCSI in3the presence or absence of heparin - 10 Ml was added, an$l H-thymidine incorporation was (10 measured. The results (mean - sem) of three experiments done in triplicates were expressed as percentages of proliferation.
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1988
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DISCUSSION As previously shown in our laboratory for HUVE cells (Barzu et 1985) heparin bound to HOME cells in a saturable manner. When al, the binding data were analysed according to Scatchard (1949), it was shown that the dissociation constant was five times lower for HOME cells (KD of 0.023 yM) than the KD reported previously (Barzu et al, 1985) for high affinity binding sites of HUVE cells (KD of 0.12 PM). In contrast to HUVE cells, where two classes of heparin binding sites have been demonstrated, heparin bound to HOME cells via a single class of high affinity binding sites. The number of hepari -8 binding sites were found to be lower (3 X 10 for HOME cells sites/cell) than that reported for HUVE cells (1.16 X 10 binding sites/cell). Since heparin is heterogenous in its molecular weight (Mr of 5.000 - 30.000 d for the heparin used in the study) Scatchard analysis of the binding data gives only an Tygrage number of binding sites. Comparison of the binding data of I-heparin binding for HOME cells to that obtained by Castellot et al (1985) o-% smoott muscle cells (single class of binding sites, RD = 10 M, 10 sites/cell) indicates lower binding constants for hepari y2ginding to smooth muscle cells than to HOME cells. Thus, the I-heparin binding to HOME cells in terms of affinity and binding sites seems to be intermediate to that of smooth muscle cells and HUVE cells. Microvasculature, which represents 95 % of the total vascular surface, contains only 5 % of the entire blood volume which moves relatively slowly in this compartment. The high affinity of heparin for HOME cells may, therefore, have great implications for the pharmacokinetic of heparin in vivo. Much lower affinity was observed with low molecular weight heparin and no binding occurred with chondroitin sulfate, SJAMP which possess a high degree of sulfation et al., 1980) competed strongly and a high molecular weight (Fan with heparin binding to HOME cells. As demonstrated for HUVE cells (Barzu et al, 1985, 1986), the heparin binding for HOME cells seems to depend on the molecular weight and the degree of sulfation of the As found for other vascular cells (Barzu et glycosaminoglycans. 1985), there was only a partial al., 1985 ; Castellot et al., reversibility of the heparin binding at 37'C suggesting that a fraction of bound heparin (23.8 X) was internalized. Heparin seems to modulate the growth of endothelial cells. It inhibits the proliferation of HUVE cells at low concentration of 1986) and the proliferation of human serum (Rosenbaum et al., bovine capillary cells induced by HBGF-I and II (Gospodarowicz et al., 1986a). In contrast, it enhances mitogenesis in HUVE cells induced by HBGF-I (ECGF/aFGF) (Gospodarowicz et al., 1986a ; Maciag study, we et al., 1984 ; Schreiber et al., 1985). In the present have investigated heparin and SJAMR on the the activity of proliferation of HOME cells and compared the effect on DNA synthesis of exponentially growing HOME cells to that of HUVE cells. Heparin inhibited growth of HOME cells induced by 5 % FCS as measured by cell counting. In log-phase growing HOME cells and HUVE cells, heparin and SJAMP reduced DNA synthesis. The inhibitory effects of heparin and SJAMP on DNA synthesis was found to be of the same magnitude for HOME cells and for HUVE cells. This does not correlate with the differences in the affinities of both cell types for
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1988
heparin. It has been previously shown for HUVE cells (Rosenbaum et weight heparin fragments retain a al, 1986) that low molecular similar inhibitory capacity than standard heparin in the presence of serum despite their lower binding affinities for HUVE cells. Thus, differences in the heparin binding affinities for endothelial cells may not imply differences in the inhibitory effects of heparin on endothelial cell proliferation. The reason for this is presently unknown. DNA synthesis of serum-deprived HOME cells was not inhibited by heparin. This may be in relation with different cellular densities in the log-phase and serum-deprived assay. Contradistinctive growth responses have also been observed for the effect of EGF on rat intestinal epithelial cells (Blay and Brown, 1987) and the effect of transforming growth factor on smooth muscle cells (Majak, 1987) where the proliferation of cells is inhibited in the exponential growth phase, but stimulated when cells reach high cellular densities. A conceptual framework regarding the effect of heparin on cell Several studies indicate that proliferation is not yet established. interfere with the binding of growth heparin binds to growth factors, and modify the internalization of the factors to their receptor, heparin biosynthesis. In endothelial cells, receptor or its stabilizes the tertiary structure of HBFG-I (ECGF/aFGF) (Schreiber 1986b) and increases its et al., et al., 1985 ; Gospodarowicz binding to the cell surface with exception of the brain derived growth factor for which the binding to bovine aortic endothelial cells is inhibited by heparin (Huang et al., 1985). In contrast, it inhibits the binding of HBGF-II (Moscatelli, 1987). In smooth muscle Reilly et al. (1987) have demonstrated that heparin inhibits cells, This the binding of EGF, but does not block the PDGF binding. inhibition is related to a decrease of EGF receptors induced by an intracellular action of heparin. In the present study, heparin exhibited an inhibitory effect on cell proliferation in the absence of HBGF-I and HBGF-II. The possibility that this effect is mediated by an intracellular mechanism as observed for smooth muscle cells is under investigation. Acknowledgements This work was supported by a grant from the "deutsche gemeinschaft" (No BI 332-l) for A.B.
Forschungs-
REFERENCES BARZU, T. , MOLHO, P., TOBELEM, G., PETITOU, M., and CAEN, J. (1985) Binding and endocytosis of heparin by human endothelial cells in culture. Biochem. Biophys. Acta, 845, 196-203 PETITOU, M., MOLHO, P., TOBELEM,G., BARZU, T., VAN RIJN, J.L.M.L., and CAEN, J. (1986) Endothelial binding sites for heparin. Biochem. J., 238, 1085-1097 BLAY, J. and BROWNK.D. (1986) Contradistinctive growth responses of cultured rat intestinal epithelial cells to epidermal growth factor depending on cell population density. J. Cell. Physiol., 129, 343-346
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CASTELLOT, J.J.L., FAVREAU, V., KARNOVSKY, M.J., and ROSENBERG, R.D. (1982) Inhibition of smooth muscle cell growth by endothelial cell-derived heparin : possible role of platelet endoglycosidase. J. Biol. Chem., 257, 11256-11260 CASTELLOT, J., WONG, K., HERMAN, B., HOOVER, R., ALBERTINI, D., WRIGHT, T., CALEB, B., and KARNOVSKY, M. (1985) Binding and internalization of heparin by vascular smooth muscle cells. J. Cell. Physiol., 124:13-20 FAN H.Z., CHEN J.D., and LIN, K.Z. (1980) An acidic mucropolysaccharide isolated from stipocus japonicus selenka and some of its physical and chemical properties. Yao Hseuh Hseuh Pao, 15, 263-270 GIMENEZ-GALLEGO, G., CONN, G., HATCHER, V.B., and THOMAS, K.A. (1986) Human brain derived acidic and basic fibroblast growth factors : amino terminal sequence and specific mitogenic activities. Biochem. Biophys. Res. Comm., 135, 541-54g5 GREENWOOD,F.C., HUNTER, W.H., and GLOVER, J.S. (1963) I-labeled growth hormone of high specific radioactivity. Biochem. J., 89, 114-123 GOSPODAROWICZ,D., MASSOGLIA, S., CHENG, J., and FUIJJI, D.K. (1986a) Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex an corpus luteum capillaries. J. Cell. Physiol., 127, 121-136 GOSPODAROWICZ,D. and CHENG, J. (1986b) Heparin protects basic and acidic FGF from inactivation. J. Cell. Physiol., 128, 475-484 HUANG, J.S., HUANG, S.S., and KUO, M.D. (1985) Bovine brain derived growth factor. Purification and characterization of its interaction with responsive cells. J. Biol. Chem., 260, 11600-11067 JAFFE, E.A., NACHMAN, R.L., BECKER, C.G., and MINICK, CV.R. (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest., 52, 2745-2756 and culture KERN, P., KNEDLER, A., and ECKEL, R.H. (1983) Isolation of microvascular endothelium from human adipose tissue. J. Clin. 71, 1822-1829 Invest., MACIAG, T., MEHLMAN, T. FRIESEL, R., and SCHREIBER, A.B. (1984) Heparin binds endothelial cell growth factor the principal endothelial mitogen in bovin brain. Science, 225, 932-935 MAJAK, R.A., and CLOWES, A.W. (1984) Inhibition of vascular smooth muscle cell migration by heparin-like glycosaminoglycans. J. Cell. Physiol., 118, 253-256 MOSCATELLI, D. (1987) High and low affinity binding sites for basic fibroblast growth factor on cultured cells : absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells. J. Cell. Physiol., 131, 131-140 ROSENBAUM, J., TOBELEM, G., MOLHO, P., BARZU, T., and CAEN, J. (1986) Modulation of endothelial cell growth by heparin. Cell Biol. Int. Rep., 10, 437-446
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7988
REILLY, C.F., FRITZE, L., and ROSENBERG,R.D. (1987) Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor. J. Cell. Physiol., 131, 149-157 SCATCHARD,G. (1949) The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci., 51, 660-672 SCHREIBER, A.B., KENNEY, J., KOWALSKI, W.J., FRIESEL, R., MEHLMAN, T.V. and MACIAG, T. (1985) Interaction of endothelial cell growth by receptor and antibody factor with heparin : characterization recognition. Proc. Natl. Acad. Sci. USA, 82, 6138-6142 TERRANOVA,V.P., DIFLORIA, R., LYALL, R.M., HIC, S., FRIESEL, R. and MACIAG, T. (1985) Human endothelial cells are chemotactic to endothelial cell growth factor and heparin. J. Cell. Biol., 101, 2330-2334 VANNUCHI, S., PASQUALI, F., CHIARUGI, V., RUGGIERO,M. (1986) Internalization and metabolism of endogenous heparin by cultured endothelial cells. Biochem. Biophys. Res. Comm., 140, 294-301
Received
26.8.88.
Accepted
29.9.88