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Circulation. 1997;95:1853-1862

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(Circulation. 1997;95:1853-1862.)
© 1997 American Heart Association, Inc.

Articles

Endogenous Basic Fibroblast Growth Factor Displaced by Heparin From the Lumenal Surface of Human Blood Vessels Is Preferentially Sequestered by Injured Regions of the Vessel Wall

Benjamin Medalion, MD; Gideon Merin, MD; Helena Aingorn, PhD; Hua-Quan Miao, MD; Arnon Nagler, MD; Amir Elami, MD; Rivka Ishai-Michaeli, MSc; Israel Vlodavsky, PhD

From the Departments of Thoracic and Cardiovascular Surgery (B.M., G.M., A.E.), Oncology (H.A., H.-Q.M., R.I.-M., I.V.), and Bone Marrow Transplantation (A.N.), Hadassah-Hebrew University Hospital, Jerusalem, Israel.

Correspondence to Dr Israel Vlodavsky, Department of Oncology, Hadassah Hospital, POB 12000, Jerusalem, 91120, Israel.


*    Abstract
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 *Abstract
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Background Proliferation of smooth muscle cells (SMCs) of the arterial wall in response to local injury is an important factor in vascular proliferative disorders. Among the growth factors that promote SMC proliferation is basic fibroblast growth factor (bFGF), which is characterized by a high affinity for heparin and is associated with heparan sulfate on cell surfaces and extracellular matrices. We investigated whether heparin can displace endogenous active bFGF from the lumenal surface of blood vessels, whether bFGF is preferentially bound to injured blood vessels, and whether a synthetic, polyanionic, heparin-mimicking compound (RG-13577) can prevent sequestration of bFGF by the vessel wall.

Methods and Results Injured and noninjured saphenous vein segments were perfused with or without heparin, in the absence or presence of 125I-bFGF and/or RG-13577 (a polymer of 4-hydroxyphenoxy acetic acid). Heparin displaced bFGF from the lumenal surface of the vein, and the released bFGF stimulated proliferation of SMCs. Likewise, systemic administration of heparin during open heart surgery resulted in a marked increase in plasma bFGF levels. Injured veins sequestered 125I-bFGF to a much higher extent than noninjured vein segments, both in the absence and presence of heparin. This sequestration was inhibited by compound RG-13577.

Conclusions Despite its beneficial effects, heparin may displace active bFGF, which subsequently may be preferentially deposited on injured vessel walls, thus contributing to the pathogenesis of restenosis. This effect may be prevented by a synthetic heparin-mimicking compound.


Key Words: heparin • growth inhibitory substances • veins • cells, smooth muscle


*    Introduction
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 *Introduction
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Proliferation of vascular SMCs with subsequent intimal thickening is a major event in neointimal formation following balloon angioplasty, endarterectomy, and CABG, especially when vein grafts are applied as conduits.1 2 3 4 Under physiological conditions most arterial SMCs remain in the G0 phase. Following EC injury, however, platelet- and nonplatelet-derived growth-promoting factors are released and initiate SMC migration and proliferation.1 2 4 5 Among these growth factors is bFGF (also known as FGF-2), a potent heparin-binding mitogen for both SMCs and ECs6 7 8 that plays a role in the pathogenesis of the initial fibromuscular formation that causes vascular grafts to stenose.9 10 11 bFGF appears to be stored as a complex with HS proteoglycans in basement membranes and extracellular matrices in vivo.12 13 14 Expression of bFGF in normal human tissues has been ubiquitously detected in basement membranes of blood vessels,15 and it is also found on the apical surface of cultured vascular ECs.16 Heparin/HS–bound bFGF is stabilized and protected against proteolytic degradation17 and can be released in an active form by heparin-like molecules18 and HS-degrading enzymes.19

Recent studies on the mode of action of bFGF have identified a novel role for heparin/HS in the formation of distinct bFGF-heparin/HS complexes that are involved in the binding of bFGF to its high-affinity tyrosine kinase receptor.20 21 22 Polyaromatic heparin-mimicking anionic molecules compete with heparin/HS for bFGF binding and hence inhibit bFGF receptor binding and mitogenic activity.23 24 25 Studies on the fate of intravenously administered 125I-bFGF show that bFGF is sequestered by the lumenal surface of the vascular endothelium and is accessible for release by heparin.18 26 27 Moreover, intravenous administration of heparin leads to a rapid increase in FGF-like activity in the circulation of rabbits.27 Because heparin is widely used as an anticoagulant, it is conceivable that it will displace bFGF from the lumenal surface of the vascular endothelium. The circulating heparin/FGF complexes may then be accessible to injured regions of blood vessels wherever they exist and stimulate SMC proliferation and migration. In the present work we used human saphenous vein segments to investigate (1) whether heparin can displace endogenous and mitogenically active bFGF from the lumenal surface of blood vessels, (2) whether heparin/bFGF complexes are preferentially sequestered by injured blood vessel segments, and (3) whether a synthetic polyanionic heparin-mimicking compound can prevent sequestration of the released bFGF by the vessel wall.


*    Methods
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 *Methods
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Materials
Recombinant human bFGF was kindly provided by Takeda Chemical Industries. Affinity-purified neutralizing anti-bFGF antibodies were kindly provided by Dr G. Neufeld, Department of Biology, Technion, Haifa.16 Sodium heparin from porcine intestinal mucous (PM-heparin, Mr=14 000, anti–factor Xa 165 IU/mg) was obtained from Hepar Industries. Bacterial (Flavobacterium heparinum) heparinase I (EC 4.2.2.7) was kindly provided by Dr J. Zimmermann, IBEX Technologies, Montreal, Canada. Compound RG-13577, a polymer of 4-hydroxyphenoxy acetic acid (Mr{approx}5800), was synthesized and kindly provided by Drs John Regan and Michael Chang, Rhone-Poulenc Rorer Co, Collegeville, Pa.23 Computer-assisted molecular modeling suggests that compound RG-13577 forms a superstructure that ensures a regular spatial distribution of negative charges, similar to that of heparin. DMEM (1 or 4.5 g glucose/L), FCS, calf serum, penicillin, and streptomycin were obtained from GIBCO. Saline containing 0.05% trypsin, 0.01 mol/L sodium phosphate, and 0.02% EDTA (STV) was obtained from Biological Industries. Tissue-culture dishes and multiwell plates were obtained from Nunc. [3H]thymidine and Na125I were obtained from Amersham. All other chemicals, which were of reagent grade, were purchased from Sigma.

Cells
SMCs were isolated from bovine aortic media.28 29 Briefly, the abdominal segment of the aorta was removed, and the fascia was cleaned away under a dissecting microscope. The aorta was cut longitudinally, and small pieces of the media were carefully stripped from the vessel wall. Two or three such strips with average dimensions of 2x2 mm were placed in 60-mm tissue-culture dishes that contained DMEM (4.5 g glucose/L) supplemented with 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Within 7 to 14 days, large patches of multilayered cells migrated from the explants. Approximately 1 week later, the cells were subcultured in 100-mm tissue-culture plates (4 to 6x105 cells/plate). The cultures (passages 3 through 8) exhibited a morphology that was characteristic of vascular SMCs, and the cells were specifically stained with monoclonal antibodies that selectively recognize the muscle form of actin (HHF-35). This antibody does not recognize ECs or fibroblasts.30 Clonal populations of bovine aortic ECs were established and cultured in DMEM (1 g glucose/L) supplemented with 10% calf serum.8 31 32 BALB/c 3T3 cells were maintained in DMEM (4.5 g glucose/L) supplemented with 10% FCS, 50 U/mL penicillin, and 50 µg/mL streptomycin at 37°C in a 10% CO2 humidified incubator.32

Growth Factor Activity
DNA synthesis in 3T3 cells was assayed.32 Briefly, BALB/c 3T3 cells were seeded at half confluence in 0.3-cm2 microtiter wells in DMEM (4.5 g glucose/L) supplemented with 10% FCS. Four to six days after reaching confluence, the medium was replaced by DMEM containing 0.2% FCS, and the cultures were further incubated for 48 hours. Samples and [3H]thymidine (5000 mCi/mmol; 1 µCi/well) were then added to the quiescent cells, and after an incubation of 32 to 40 hours, DNA synthesis was assayed by measuring the radioactivity incorporated into trichloroacetic acid–insoluble material. For measurements of EC proliferation, cells were seeded at a low density (1x103/16-mm culture well) in 1 mL DMEM containing 10% heat-inactivated calf serum. bFGF or aliquots (10 to 20 µL) of the perfusates were added to some of the wells on days 2 and 4. Six days after seeding, the cells were dissociated with STV and counted in a Coulter counter (Coulter Electronics, Ltd).8 SMCs were plated (4x104 cells/16-mm well) in DMEM supplemented with 10% FCS. Twenty-four hours after seeding, the medium was replaced with medium containing 0.2% FCS, and 48 hours later the cells were exposed to growth stimulants and [3H]thymidine (1 µCi/well) for an additional 24 to 48 hours. DNA synthesis was assayed by measuring the radioactivity incorporated into trichloroacetic acid–insoluble material.25 Each experiment was performed in triplicate at least three times; the variations between different experiments did not exceed 20%.

Iodination and Binding of bFGF
Recombinant bFGF was iodinated by using chloramine T.24 25 Briefly, 5 µg bFGF was added to 50 µL of 0.2 mol/L sodium phosphate, pH 7.2, containing 1 mCi Na125I. Chloramine T (10 µL of 1 mg/mL) was added for 45 seconds at room temperature, and the reaction was stopped by adding 50 µL of 1 mg/mL sodium metabisulfite and 50 µL of 2 mg/mL NaI. The reaction mixture was then applied to a 0.2-mL heparin-Sepharose (Pharmacia) column equilibrated with 0.6 mol/L NaCl in 20 mmol/L phosphate buffer, pH 7.2. The column was washed with the same buffer, and the 125I-bFGF was eluted with 0.5 mL phosphate buffer containing 2 mol/L NaCl, 0.3% CHAPS, and 0.1% BSA. The specific activity was 1.2 to 1.7x105 cpm/ng bFGF, and the labeled preparation was kept for up to 3 weeks at -70°C. The iodinated bFGF yielded a single band at 18.4 kD when subjected to PAGE and autoradiography, and it retained full mitogenic activity (stimulation of [3H]thymidine incorporation) when added to growth-arrested 3T3 fibroblasts.

Displacement of bFGF by Heparin
Leftover segments ({approx}2 cm long) of human saphenous vein that were harvested from patients undergoing CABG were flushed with saline and kept in saline for <30 minutes at 24°C. Specimens were incubated for 1 hour at 24°C in either 2 mL saline or saline containing 7.5 U/mL heparin, and aliquots of the incubation media were tested for growth-promoting activity and bFGF content (Quantikine, human bFGF ELISA assay, R&D Systems). Alternatively, a saphenous vein specimen was cut transversely into two equal segments. Each segment was kept intact, cannulated at both ends with perfusion cannula (Vessel cannula, DLP), and perfused at 24°C with saline or saline plus 7.5 U/mL heparin for 15 minutes at a flow rate of 2 mL/min. Aliquots of the perfusates were tested for mitogenic activity.

Sequestration of 125I-bFGF
A saphenous vein specimen was cut into two equal segments as described above. One segment was left intact, and the other was subjected to mechanical damage by gently scratching the inner surface of the vein with forceps (Adson forceps, Rush-Pilling SA). Each of the vein segments was perfused for 15 minutes at 2 mL/min at 24°C with 2 mL bFGF-binding medium (DMEM [4.5 g glucose/L], 25 mmol/L HEPES, pH 7.5, and 0.1% BSA) and 125I-bFGF (5 ng/mL) in the absence or presence of either heparin (7.5 U/mL), compound RG-13577 (7.5 µg/mL), or both. In some experiments the vein segments were pretreated (30-minute perfusion at 24°C) with bacterial heparinase (0.15 U/mL saline containing 0.5 mmol/L CaCl2), washed with saline ({approx}50 mL), and perfused with 125I-bFGF as described above. To measure the amount of 125I-bFGF sequestered by the inner surface of the vein, the vein segments were perfused with saline for 15 minutes at 24°C, cut longitudinally, weighed, and counted in a gamma counter. LAB 125I-bFGF was then eluted with 25 mmol/L HEPES, pH 7.4, containing 2 mol/L NaCl, and both the eluate and vein segments were counted in a gamma counter. HAB bFGF was determined by a subsequent 5-minute incubation in a solution containing 2 mol/L NaCl and 20 mmol/L sodium acetate, pH 4.5.33 34 Both the incubation medium and vein segments were then counted in a gamma counter. Binding data are expressed as nanograms of bFGF per gram of vein tissue.

Quantification of bFGF Levels During CPB
Blood samples were taken in buffered sodium citrate solution at the beginning of CABG before the patient received heparin, 30 minutes after heparinization, when the patient was fully heparinized, and at the end of the operation. The blood was centrifuged for 10 minutes at 1500g at 4°C, and aliquots of the plasma were tested for bFGF content by using an ELISA assay as described above. The sensitivity of the assay is 1.0 pg/mL; the dynamic range for plasma samples is 10 to 640 pg/mL. There was no cross-reactivity or interference with bFGF-related or unrelated cytokines. A standard curve was included in each ELISA assay to control for the sensitivity, linearity, and reproducibility of the assay. Each plasma sample was tested in triplicate, and both the intra- and intervariation between different determinations did not exceed ±5%.

Statistical Analysis
Each experiment was performed at least three times, and either triplicate wells (cell proliferation; ELISA tests) or single vein segments (125I-bFGF binding) were used in each experiment. Means and SDs were calculated. The data presented in Figs 1Down, 2Down, and 6Down (cell proliferation) and Fig 3Down (ELISA) were subjected to single-factor ANOVA (P<.05). In addition, a multiple-comparison post hoc test model was applied. A two-tailed, two-sample Student's t test for differences in means was performed for the means of all groups. Each of the t tests evaluated the difference between the means of two of the groups in the model. Each comparison t test had a calculated significance level of .005; the significance adjusted for all comparisons in the model was .05. Comparisons of 125I-bFGF binding to pairs of intact and injured vein segments (Figs 4Down, 5Down, and 7Down) under each experimental condition were performed by using an unpaired, two-tailed Student's t test. Significance was defined as P<.05. Statistical analysis of the results was performed with the StatWork package (Cricket Software, Inc).



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  		Figure 1. Bar graph. Saphenous vein segments (each {approx}2 cm long) were perfused for 15 minutes at 24°C (hatched bars) with saline (vein+saline) or saline containing 7.5 U/mL heparin (vein+heparin). Aliquots of the perfusates were tested for stimulation of [3H]thymidine incorporation in growth-arrested 3T3 fibroblasts in a 96-well assay (0.2 mL/well) as described in "Methods." Saline (20 µL), heparin (7.5 U/mL), or recombinant bFGF (0.1 ng/mL) (open bars) was added directly to some wells as a control. The heparin perfusate and recombinant bFGF were also tested for growth-promoting activity in the presence of neutralizing rabbit anti-bFGF IgG (75 µg/mL) (solid bars). Each point represents mean±SD of triplicate wells; variations between different experiments did not exceed 20%.



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  		Figure 2. Bar graphs. Saphenous vein segments were perfused for 15 minutes at 24°C with saline (vein+saline) or saline containing 7.5 U/mL heparin (vein+heparin). Aliquots of the perfusates were tested for stimulation of (A) [3H]thymidine incorporation in growth-arrested vascular SMCs and (B) vascular EC proliferation as described in "Methods." Saline, heparin (final concentration, 0.75 U/mL), or recombinant bFGF (final concentration, 0.5 ng/mL) was added as a control. Each point represents mean±SD of triplicate wells.



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  		Figure 6. Bar graph. SMCs (4x104 cells/well) were seeded into 24-well plates in medium containing 10% FCS. Twenty-four hours after seeding, the cells were arrested by a 48-hour incubation in medium containing 0.2% FCS. The SMCs were incubated for an additional 48 hours in the presence of 1 µCi/0.5 mL [3H]thymidine in the absence (-FGF) or presence (+FGF) of 1 ng/mL bFGF without (open bars) or with (hatched bar) 5 µg/mL heparin, 5 µg/mL RG-13577 (solid bar), or both (cross-hatched bar). Thymidine incorporation was determined as described in "Methods." Each point represents mean±SD of triplicate wells.



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  		Figure 3. Bar graph. Blood samples (5 mL) were taken in buffered sodium citrate at the beginning of CABG before the patient received heparin, 30 minutes after heparinization, and 160 minutes later (just before the patient left the operating room). Plasma levels of bFGF were determined by using an ELISA as described in "Methods." Error bars represent the SD of mean plasma bFGF levels as determined in 11 patients. bFGF levels 30 minutes after heparinization were higher (P<.005) than those before heparinization and at the end of surgery. Variations between triplicate determinations of the ELISA did not exceed ±5% of the mean.



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  		Figure 4. Bar graphs. A, Saphenous vein specimens were cut into two equal segments. One segment was left intact (open bars), and the other was subjected to mechanical damage (hatched bars). Each segment was perfused for 15 minutes (2 mL/min) at 24°C with bFGF-binding medium and 5 ng/mL 125I-bFGF followed by a 10-minute perfusion with saline and counting in a gamma counter (vein after perfusion). LAB 125I-bFGF was released by a 5-minute exposure to 2 mol/L NaCl, pH 7.4, and both the incubation medium (LAB) and vein tissue (vein after LAB) were subjected to gamma counting. HAB 125I-bFGF was then released by a 5-minute incubation with 2 mol/L NaCl, pH 4.5, and both the incubation medium (HAB) and vein tissue (vein after HAB) were counted in a gamma counter. B, The experiment was performed as described in A except that 7.5 U/mL heparin was included in the perfusion solution. Each bar represents mean±SD of three experiments; data are expressed as nanograms 125I-bFGF bound per gram of vein tissue. P<.05 injured vs corresponding noninjured samples.



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  		Figure 5. Bar graph. A saphenous vein specimen was injured and cut into two segments. One segment was perfused for 15 minutes (2 mL/min) at 24°C with bFGF-binding medium containing 5 ng/mL 125I-bFGF (cross-hatched bars), and the other segment was perfused with the same medium but in the presence of 7.5 µg/mL compound RG-13577 (solid bars). The amount of 125I-bFGF bound to the vein immediately after perfusion (vein after perfusion) and the amounts of 125I-bFGF released and remaining bound to the vein after exposure to 2 mol/L NaCl, pH 7.4 (LAB and vein after LAB), and 2 mol/L NaCl, pH 4.5 (HAB and vein after HAB), were determined as described in the legend to Fig 4Up. Each bar represents mean±SD of three experiments; data are expressed as nanograms 125I-bFGF bound per gram of vein tissue. P<.05 bFGF binding in the absence vs presence of RG-13577.



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  		Figure 7. Bar graph. A saphenous vein specimen was injured and cut into two segments. One segment was pretreated for 30 minutes at 24°C with 0.15 U/mL bacterial heparinase, followed by perfusion for 15 minutes (2 mL/min) at 24°C with 5 ng/mL 125I-bFGF (solid bars). The other segment was first perfused with saline alone, followed by perfusion with 125I-bFGF (cross-hatched bars). The amounts of 125I-bFGF bound to the vein immediately after perfusion (vein after perfusion), LAB, vein after LAB, HAB, and vein after HAB were determined as described in the legend to Fig 4Up. Each bar represents mean±SD of three experiments; data are expressed as nanograms 125I-bFGF bound per gram of vein tissue. P<.05 bFGF binding with vs without heparinase treatment.


*    Results
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 up arrowMethods
 *Results
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Heparin Displaces Active bFGF From the Lumenal Surface of Saphenous Vein
Segments (each 2 cm long) derived from the same saphenous vein during a CABG operation were cut longitudinally and incubated for 1 hour at 24°C with saline or saline containing 7.5 U/mL heparin (final volume, 2 mL each). Aliquots (15 µL) of the incubation media were tested for mitogenic activity on growth-arrested 3T3 fibroblasts. A high growth-promoting activity was exerted only by the heparin-containing incubation medium, and there was little or no activity after incubation of the vein with saline alone (not shown). Heparin alone failed to exert any mitogenic effect. In subsequent experiments, the vein was kept intact, cannulated at both ends, and perfused with 2 mL saline with or without heparin for 15 minutes at a flow rate of 2 mL/min. This procedure assured that the mitogenic activity was derived exclusively from the lumenal surface of the vein and not from the adventitia and/or the cut edges of the vein segment. A high mitogenic activity toward 3T3 fibroblasts, similar to that induced by 0.1 ng/mL recombinant human bFGF, was induced by the heparin perfusate (Fig 1Up, vein+heparin) compared with a very low activity observed when the vein was perfused with saline (Fig 1Up, vein+saline). Induction of DNA synthesis in 3T3 fibroblasts by the heparin perfusate was dose dependent (not shown). Affinity-purified neutralizing anti-bFGF antibodies16 inhibited the growth-promoting activity of the heparin perfusate by 85% to 95%.

To further characterize the nature of the growth-promoting activity displaced by heparin from the lumenal surface of the vein, we tested the activity of the perfusates on vascular SMCs (Fig 2AUp) and ECs (Fig 2BUp). In both cell types, mitogenic activity was induced by the heparin perfusate (vein+heparin) but not by the saline perfusate. Thus, a two- to threefold increase in DNA synthesis was induced in growth-arrested SMCs by 10 µL of the heparin perfusate compared with the saline perfusate, similar to the effect of 0.5 ng/mL bFGF (Fig 2AUp). In fact, there was no detectable activity in the saline perfusate above the level of thymidine incorporation by the resting SMCs when no perfusate was added (Fig 2AUp, saline). Similar results were obtained with vascular ECs (Fig 2BUp). While heparin alone slightly inhibited cell growth, a sevenfold increase in cell number was obtained in the presence of the heparin perfusate (Fig 2BUp, vein+heparin versus heparin), slightly less than the effect of exogenously added recombinant bFGF (0.5 ng/mL). A slight (20% to 30%) stimulation of EC proliferation was induced by the saline perfusate. Unlike the experiments with the 3T3 fibroblasts and SMCs, which were growth arrested, the ECs were exposed to the perfusates in the presence of 10% calf serum, suggesting the involvement of a growth factor (ie, bFGF) that is not found in serum.

To better identify and quantify the amount of bFGF extracted from the vein during perfusion, aliquots of the heparin and saline perfusates were subjected to a specific human bFGF ELISA immunoassay (Quantikine, human bFGF, R&D systems). The heparin perfusate contained 119±11 pg/mL bFGF; the saline perfusate, 21±2 pg/mL bFGF (P<.05). It can therefore be concluded that heparin displaces active bFGF from the lumenal surface of the vascular endothelium, which may then stimulate proliferation of vascular ECs and SMCs.

Effect of Heparin Administration on bFGF Levels in Human Plasma
The levels of bFGF in the plasma of patients undergoing CABG were determined by using an ELISA. For this purpose, blood samples from 11 patients were taken at the beginning of the operation, before the patient received heparin, 30 minutes after the beginning of CPB (when the patient was fully heparinized), and at the end of the operation. Significant increases (three- to fivefold in most patients and up to 10-fold in some patients; P<.005 by t test) in the amount of plasma bFGF were obtained during CPB (when the patient was under heparin) compared with the levels of bFGF determined either before the administration of heparin or at the end of the operation (Fig 3Up). The increase in plasma bFGF was detected within minutes after heparin injection, reached a maximum level within 15 minutes, and decreased toward the end of the operation, ie, 2 to 3 hours after heparinization. These results indicate that circulating heparin may displace relatively high amounts of bFGF ({approx}40 pg/mL and up to {approx}100 pg/mL) from the surface of the vascular endothelium during CPB. Concentration of plasma samples on heparin-Sepharose followed by salt ({approx}1.7 mol/L) elution revealed that the heparin-displaced factor was highly active (3T3 assay, not shown), similar to results obtained in rabbits.27

Sequestration of 125I-bFGF by Normal Versus Injured Regions of Saphenous Veins
bFGF exerts its mitogenic activity through both high-affinity tyrosine kinase signaling receptors and low-affinity HS accessory receptors on the cell surface.7 20 21 To compare sequestration of bFGF by noninjured and injured regions of human saphenous vein, veins were cut into two segments. One segment was left intact, and the other was subjected to mechanical damage by gently scratching the inner surface of the vein with a forceps. Each of the vein segments was washed with saline and perfused with 125I-bFGF, and the amounts of LAB and HAB bFGF were determined.33 34 As shown in Fig 4AUp (vein after perfusion), the amount of 125I-bFGF bound to injured vein segments was 5.3±1.8-fold higher than that sequestered by noninjured segments. A similar result was obtained when the vein segments were washed and briefly incubated with 2 mol/L NaCl at pH 7.4 (LAB) or pH 4.5 (HAB) (Fig 4AUp). Three- to fivefold (3.7±0.7) more bFGF remained bound per gram of injured than noninjured vein tissue even after the final exposure of the veins to 2 mol/L NaCl, pH 4.5, which was applied to release the 125I-bFGF from high-affinity cell-surface receptor sites (Fig 4AUp). Altogether these results indicate that bFGF is sequestered by damaged regions of the vessel wall to a much larger extent than by noninjured regions. Heparin displaces 125I-bFGF and bFGF-like activity from the subendothelial extracellular matrix18 and the lumenal surface of blood vessels,18 26 27 which suggests that bFGF binds to heparin with a higher affinity than to HS. Sequestration of 125I-bFGF by noninjured and injured vein segments was markedly reduced (66±7.1%) in the presence of 7.5 U/mL heparin during the perfusion. Despite this inhibitory effect of heparin, the difference in bFGF binding capacity between the noninjured and injured vein segments was retained (injured/noninjured=3.5±1) (Fig 4BUp, vein after perfusion).

Vessel Wall Sequestration of 125I-bFGF Is Prevented by a Synthetic `Heparin-Mimicking' Compound and by Pretreatment With Heparinase
A series of negatively charged, nonsulfated polyaromatic compounds mimic many of the effects of heparin.23 24 25 These nontoxic compounds compete for bFGF binding to HS on the cell surface and extracellular matrix.24 25 We investigated whether the application of one of these compounds, RG-13577 (a polymer of 4-hydroxyphenoxy acetic acid),25 could abolish the sequestration of bFGF by the vessel wall. For this purpose, injured vein segments were perfused with 125I-bFGF in the absence or presence of 7.5 µg/mL compound RG-13577, and levels of LAB and HAB bFGF were determined. An almost complete inhibition (86.5±4.4%) of 125I-bFGF deposition was obtained in the presence of RG-13577 (Fig 5Up). Both low- and high-affinity binding of 125I-bFGF were prevented under this condition, indicating that compound RG-13577 is a highly efficient inhibitor of bFGF deposition on the inner surface of injured vessels. It should be noted that vessel wall sequestration of 125I-bFGF was also inhibited in the presence of heparin (Fig 4BUp), but to a lesser extent than in the presence of compound RG-13577 (Fig 5Up). When both heparin and RG-13577 were included in the perfusion solution, binding of 125I-bFGF was reduced to the level observed in the presence of RG-13577 alone (not shown), indicating that compound RG-13577 can exert its inhibitory effect in the presence of heparin. We have reported that compound RG-13577 is also a potent inhibitor of vascular SMC proliferation.25 This was also demonstrated in the present study by showing that RG-13577, either alone or in the presence of heparin, efficiently inhibited the growth-promoting activity of bFGF on vascular SMCs (Fig 6Up). Under the same conditions there was no inhibition or even a slight stimulation of SMC proliferation by heparin (Fig 6Up). These results further emphasize the advantage of using compound RG-13577 to abolish both bFGF deposition and growth-promoting activity.

In subsequent experiments, vein segments were treated with bacterial heparinase to further elucidate the involvement of HS in bFGF sequestration by the inner surface of the vein. Pretreatment of cells and extracellular matrix with bacterial heparinase markedly reduces binding of 125I-bFGF to low-affinity receptor sites due to an efficient degradation and removal of HS side chains.35 Fig 7Up demonstrates that pretreatment (30-minute perfusion at 24°C) of an injured vein segment with heparinase I (0.15 U/mL) followed by perfusion with 125I-bFGF resulted in a marked inhibition of bFGF sequestration (125I-bFGF binding in the absence versus presence of heparinase treatment=3.53±0.9). This result further demonstrates that bFGF is sequestered by the lumenal surface of the vein primarily through binding to HS moieties.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study demonstrated that heparin administration in human patients is associated with an increased level of circulating plasma bFGF. While the mean level of plasma bFGF is normally <10 pg/mL, the bFGF levels (as determined by ELISA) in patients who received heparin during CPB were markedly increased, reaching a concentration of {approx}40 pg/mL in most patients and up to 100 pg/mL in some patients. Using human saphenous vein segments, we demonstrated that this increase in plasma bFGF is primarily due to release of bFGF from the lumenal surface of the vessel. Moreover, the released bFGF was active in promoting the proliferation of growth-arrested 3T3 fibroblasts as well as vascular ECs and SMCs. The growth-promoting activity released from the lumenal surface of the vessel wall can be attributed primarily to bFGF, since it was almost completely inhibited in the presence of neutralizing anti-bFGF antibodies. We have shown16 that bFGF is found as a complex with HS on the surface of cultured vascular ECs and SMCs and can be released by HS-degrading enzymes and by glycosylphosphatidylinositol-specific phospholipase C.

Animal studies in which recombinant 125I-bFGF has been administered intravenously reveal that the bFGF is immediately sequestered by the inner surface of the vessel wall and can be displaced upon injection of heparin.18 26 27 In these experiments, heparin slowed the rate of bFGF clearance and prolonged the half-life of plasma bFGF, resulting in a higher plateau level of bFGF in the blood during continuous infusion.26 These results were attributed to sequestration of the exogenously added 125I-bFGF by HS on the EC surfaces and its subsequent release and stabilization by heparin.17 18 26 27 Likewise, intravenous administration of heparin causes a rapid rise in FGF-like activity in the circulation of normal rabbits.27 This result and the present results with human patients and isolated saphenous vein segments indicate that endogenous bFGF is normally associated with the inner surface of the vessel wall and can be displaced in an active form by heparin. Vascular SMCs and ECs in intact noninjured regions of the vessel wall are refractory to stimulation by circulating bFGF.26 Intravenously administered bFGF is cleared from the circulation predominantly by the liver and to a lesser extent by the kidneys, regardless of the presence or absence of heparin.18 26 Intact 125I-bFGF appears in the urine of rats receiving bFGF and heparin but not in the urine of rats receiving bFGF alone, suggesting that heparin diminishes the affinity of bFGF for HS in the glomerular basement membrane.26 The dose of heparin used in the present study (7.5 IU/mL) is similar to that given in bypass surgery, and similar doses (2 to 15 IU/mL) have been used to combat experimental restenosis in rats.36 It should be noted, however, that efficient displacement of HS-bound bFGF is already exerted at 10- to 50-fold lower concentrations of heparin.18

Inhibition of bFGF during the first 3 days after balloon injury of the rat carotid artery inhibits the first wave of SMC proliferation, indicating that bFGF plays a major role in the initial proliferative response after vascular injury.9 10 The ability of heparin to displace biologically active bFGF prompted us to investigate whether this circulating bFGF may preferentially reach injured regions of blood vessels, where it can trigger vascular SMC proliferation and neointimal formation. For this purpose, we perfused injured and noninjured human saphenous vein segments with 125I-bFGF, washed the unbound bFGF, and measured the amount of bFGF remaining firmly bound to each segment. Five- to sevenfold higher amounts of 125I-bFGF were associated with injured than with intact vessels regardless of whether binding to low- or high-affinity sites was determined. bFGF is internalized through both receptor- and HS-mediated mechanisms.37 This internalization may account for the amount of labeled growth factor that remains associated with the vein tissue after removing the 125I-bFGF bound to low- and high-affinity receptor sites. Pretreatment of the inner vessel wall with bacterial heparinase resulted in a marked inhibition of 125I-bFGF sequestration, indicating that the majority of the circulating bFGF is bound to HS on the lumenal vein surface. Vessel wall deposition of 125I-bFGF was markedly reduced by heparin. Nevertheless, higher amounts of bFGF were sequestered by injured than noninjured vein segments both in the absence and presence of heparin. It is therefore conceivable that whenever heparin is used it displaces significant amounts of bFGF ({approx}200 ng, based on a total blood volume of 5 L) from the entire vasculature and that this bFGF may then be deposited onto damaged regions of blood vessels. Such damage always occurs in areas of balloon angioplasty, endarterectomy, anastomotic sites, and along saphenous vein grafts secondary to harvesting and exposure to systemic pressure.1 2 High-pressure distension also significantly increases the number of available low- and high-affinity bFGF receptor sites in saphenous vein grafts.34

Although heparin inhibits the proliferation of SMCs in culture28 29 38 39 and intimal hyperplasia in experimental animals,36 40 it is not effective in inhibiting restenosis in humans.36 41 42 The present study raises the possibility that in spite of its antiproliferative effect, heparin may also exert an undesirable effect, ie, displacement of active bFGF that is normally bound to HS on the lumenal surface of blood vessels. The released bFGF may then be preferentially deposited on injured vessel walls, thus contributing to the pathogenesis of restenosis. Although we have demonstrated that deposition of the displaced bFGF is partially inhibited in the presence of heparin, it is well recognized that bFGF-heparin complexes are best presented to the high-affinity tyrosine kinase receptor, resulting in signal transduction and cell proliferation.20 21 22 In fact, experiments with HS-deficient cells reveal that heparin efficiently replaces the cell-surface HS and functions as a low-affinity receptor in the dual-receptor mechanism of bFGF action.20 21 22 43 Thus heparin may, on the one hand, inhibit SMC proliferation and bFGF deposition, but on the other hand it releases endogenously sequestered bFGF that may then be presented in an active form to vascular SMCs in injured regions of blood vessels. In an attempt to overcome this disadvantage of heparin we used the synthetic, polyanionic heparin-mimicking compound RG-13577 (a polymer of 4-hydroxyphenoxy acetic acid) to inhibit the sequestration of 125I-bFGF by injured vein segments. This polyanionic aromatic compound binds bFGF, but unlike heparin, it is not capable of presenting bFGF to its signaling receptor. It also prevents heparin-mediated dimerization of the growth factor and inhibits interaction of bFGF with cell-surface receptors,44 resulting in an inhibition of bFGF-mediated cell proliferation.24 25 Indeed, perfusion of an injured vein segment with 125I-bFGF in the presence of RG-13577 almost completely inhibited sequestration of bFGF, both in the absence and presence of heparin. Moreover, we have demonstrated25 that compound RG-13577 effectively inhibits SMC proliferation through a direct interaction with specific receptors on the SMC surface. In light of these considerations, administration of heparin together with compound RG-13577 or related compounds may prevent the undesirable effect of heparin mentioned above. We plan to further investigate this possibility both in vitro and in experimental animals. Alternatively, heparin may be substituted by other anticoagulants (eg, hirudin) that also inhibit intimal hyperplasia. Hirudin has been used as an anticoagulant,45 and at the same time it inhibits neointimal formation in rabbits and pigs to a higher extent than heparin.46 47 Synthetic polyanionic compounds similar to RG-13577 that exert both potent anticoagulant and antiproliferative activities have been synthesized in our laboratory and are being tested in animal models of restenosis.

Heparin/HS participates in the regulation of many cellular processes, particularly cell growth and differentiation, tumor metastasis and angiogenesis, autoimmunity, lipoprotein metabolism, and gene expression, although the mode of action has not been clearly elucidated.39 48 The antiproliferative effect of heparin has been attributed, among other possibilities, to its interaction with transcription factors and inhibition of the protein kinase C–dependent pathway that is required for the induction of c-fos and c-myc.39 49 Heparin also attenuates the induction by serum of bFGF mRNA in quiescent human saphenous vein SMCs.50 The ability of heparin to increase the bioavailability of bFGF and possibly other heparin-binding growth factors may also exert favorable effects, eg, the promotion of tissue repair processes51 and growth of collateral vessels in response to ischemia.52 53 In view of the multiple effects of heparin, natural and synthetic heparin-mimicking compounds, such as the one presented in this study, are being identified in an attempt to suppress or enhance these activities, depending on the specific clinical situation.


*    Selected Abbreviations and Acronyms
 
bFGF = basic fibroblast growth factor
CABG = coronary artery bypass graft
CPB = cardiopulmonary bypass
EC = endothelial cell
HAB = high-affinity-bound
HS = heparan sulfate
LAB = low-affinity-bound
SMC = smooth muscle cell


*    Acknowledgments
 
This research was supported by grants from the German-Israeli Foundation for Scientific Research and Development, the Israel Science Foundation (Centers for Excellence Program), the Israel Ministry of Health, and the Bundesministerium für Forschung und Technologie.

Received May 13, 1996; revision received October 8, 1996; accepted November 20, 1996.


*    References
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