Published online before print April 10, 2003, doi: 10.1161/01.RES.0000071345.76095.07
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© 2003 American Heart Association, Inc.
UltraRapid Communication |
Blockade of Vascular Smooth Muscle Cell Proliferation and Intimal Thickening After Balloon Injury by the Sulfated Oligosaccharide PI-88
Phosphomannopentaose Sulfate Directly Binds FGF-2, Blocks Cellular Signaling, and Inhibits Proliferation
From the Division of Immunology and Genetics (D.J.F., C.R.P., K.J.B., W.B.C., C.N.C.), John Curtin School of Medical Research, Australian National University, Canberra; Centre for Research in Vascular Biology (M.M., J.A.B., I.P.H., J.H.C., G.R.C.), School of Biomedical Science, University of Queensland, Brisbane; and the Centre for Thrombosis and Vascular Research (F.S.S., H.C.L., C.N.C., L.M.K.), University of New South Wales and the Department of Hematology, The Prince of Wales Hospital, Sydney, Australia.
Correspondence to Levon M. Khachigian, PhD, Associate Professor, Centre for Vascular Research, Department of Pathology, University of New South Wales, Sydney NSW 2052. E-mail L.Khachigian{at}unsw.edu.au
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Abstract |
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Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (Ki 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.
Key Words: phosphomannopentaose sulfate • heparanase inhibitor • smooth muscle cells • neointima formation • balloon angioplasty
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Phosphomannopentaose sulfate (PI-88) is a recently developed synthetic polysulfated oligosaccharide that inhibits the activity of the extracellular matrix-degrading enzyme heparanase.1 PI-88 is a 2-kDa heparan sulfate mimetic structurally distinct from heparin, a heterogenous mixture of polysaccharide chains of varying length with a molecular weights of 3 to 30 kDa. PI-88 is also structurally and functionally different from small heparin-like polyaromatic anionic-type compounds2 and other heparan sulfate mimetics.1
In vivo studies have shown that PI-88 is a potent inhibitor of tumor metastasis and angiogenesis.1 Human and mammalian toxicology studies demonstrate that PI-88 is well-tolerated and has low anticoagulant activity.3 PI-88 is currently in Phase II clinical trials to assess its therapeutic potential as an anticancer drug and is administered to patients by continuous infusion or repeated injection.
Percutaneous transluminal coronary angioplasty (PTCA) is commonly used to repair occluded atherosclerotic blood vessels. Its long-term efficacy, however, is limited by restenosis, or renarrowing of the arteries due largely to the accumulation of vascular smooth muscle cells at the site of balloon inflation. PTCA releases growth factors from the artery wall,4,5 which may act in a paracrine manner to trigger signaling and transcriptional pathways preceding proliferation, and eventually, arterial reocclusion. Antiplatelet strategies,6 and more recently, stent-based therapeutics in clinical trials7 have showed markedly reduced rates of restenosis. However, the scope of clinically proven antirestenotic agents is extremely limited, and additional strategies are needed.
In the present study, we demonstrate that PI-88 directly inhibits ERK1/2 activation in vascular smooth muscle cells within minutes of injury, inhibits smooth muscle cell proliferation, and blocks degradation of heparan sulfate, change in smooth muscle phenotype, and the formation of neointimal thickening in rat and rabbit carotid arteries after balloon angioplasty. PI-88 blocked decreased levels of FGF-2 in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering blood coagulation in these animals. Thus, PI-88 may represent a new therapeutic agent for the inhibition of arterial restenosis induced by PTCA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Drug
Phosphomannopentaose sulfate (PI-88) (Progen Industries Limited) is a sulfated oligosaccharide with a molecular weight of 2100 to 2585 and was prepared as previously described1,8 by the sulfation of phosphomannopentaose derived from the phosphomannan produced by the yeast Pichia holstii. PM5 is a minimally sulfated backbone counterpart of PI-88 with a molecular weight of 770.9
Carotid Angioplasty
Animal experimentation had prior approval from the Australian National University Animal Ethics Committee or the University of Queensland Animal Experimentation Ethics Committee. Arterial trauma and endothelial cell denudation was induced in the left common carotid artery of 16 to 20 week old male Wistar rats using as previously described.10 Briefly, the animals were anesthetized and, using aseptic surgical technique, the left common carotid artery was exteriorized through a ventral midline neck incision. A 2F Fogarty arterial embolectomy catheter (Baxter Healthcare Corporation) was inserted into the lumen of the artery in a caudal direction via an arteriotomy. The catheter was advanced for a distance of 1 cm, and the balloon distended using 0.12 mL of air so as to generate slight arterial wall resistance while the catheter was slowly withdrawn. This procedure was repeated for a total of three times after which it was removed from the arterial lumen. The artery was ligated using 5/0 silk (Ethicon), and the surgical wound closed. In a similar manner, a 2F Fogarty catheter was inserted into the right common carotid artery of 14 to 16 week old New Zealand white cross rabbits, the balloon inflated and the catheter withdrawn along the artery against resistance a total of three times to induce endothelial denudation.11
PI-88 Treatment
Immediately after completion of the carotid angioplasty in the rat, subcutaneous administration of PI-88 (in saline) was commenced at a dose of 35 mg/kg per day using 2 ML2 Alzet osmotic minipumps (Alza Corp) and continued for 2 weeks (n=6). A control group was established with osmotic minipumps containing saline only (n=7). In the rabbit, the osmotic minipumps were implanted in the peritoneal cavity and different doses were constituted to give a continuous IP infusion of saline (n=16), 0.15 (n=8), 1.5 (n=7) and 15 (n=12) mg/kg per day PI-88 over the first 7 days after carotid artery injury.
Carotid Artery Removal and Assessment
Two weeks after surgery rats were euthanized by pentobarbitone (Nembutal; Boehringer Ingelheim Pty Ltd) overdose and perfused with 10% buffered formalin for 20 minutes. The left common carotid arteries were removed between the point of Fogarty catheter insertion cranially and the aorta caudally and placed in 10% buffered formalin. A similar length of the right carotid artery was removed from each animal. The entire length of artery that had been removed was sectioned. Transverse histological sections (4 µm) were cut at multiple levels, 100 µm apart. Sections at each level were stained with Gill’s Hematoxylin No. 312 and Eosin, or Verhoeff’s Hematoxylin/Elastic stain13 to highlight the elastic lamina. Sections were examined and the cross sectional area of the neointima (from the internal elastic lamina to the lumen) and the artery (as the external elastic lamina) were determined using digital camera–generated photomicrographs and Adobe PhotoShop software (Adobe Systems Incorporated) and NIH Image software. The level of maximal intimal proliferation was determined, and the neointimal area expressed as a percentage of the neointimal area relative to the total area of the artery. Results were expressed as the mean±SEM, and n represents the number of animals in a particular group. Statistical comparisons (considered significant, P<0.05) were made with a Student’s unpaired t test. Rabbits were euthanized by Euthanal 2 weeks after surgery, and arteries perfusion fixed with 4% buffered paraformaldehyde. Transverse sections were cut at multiple levels and stained with 0.1% toluidine blue before image analysis by digital photomicroscopy. Statistical comparisons between the 4 groups were made with a one-way ANOVA on ranks.
Immunohistochemistry and Electron Microscopy
Up to 8 rabbits from the saline and PI-88 (15 mg/kg per day) groups were euthanized on day 4 and 7 after surgery without perfusion fixation of the arteries. Alternate sections of these arteries were fixed in 4% buffered paraformaldehyde or 5% glutaraldehyde in buffered saline and processed for immunohistochemistry or electron microscopy. Paraffin sections for immunohistochemistry were blocked with 0.5% casein in phosphate-buffered saline, before incubation with primary antibodies to heparan sulfate14 (10E4, Seikagaku Corp), chondroitin sulfate (CS56, Sigma), or fluorescein conjugated antibody to 
-smooth muscle actin (IA4-FITC, Sigma). The secondary antibody used was biotinylated anti-mouse IgM (Sigma), followed by streptavidin-CY5 conjugate. Slides were immediately viewed with a Bio-Rad confocal microscope system equipped with a krypton-argon laser. All settings for power, gain, and background were kept constant for viewing sections stained with each particular antibody. Tissue fixed in glutaraldehyde was processed through 1% osmium tetroxide and 5% uranyl acetate before embedding in Epon/araldite. Ultrathin sections were stained with 2% uranyl acetate and lead citrate and viewed by transmission electron microscopy for ultrastructural morphometry. Forty random, nonconsecutive photomicrographs (x8000) were taken for each artery for quantitation of the smooth muscle cell cytoplasmic volume fraction of myofilaments (Vvmyo). Statistical comparisons were made with a Student’s unpaired t test.
Smooth Muscle Cell Proliferation Assay
Primary rat aortic smooth muscle cells were growth-arrested by incubation overnight in serum-free medium (Waymouth’s, pH 7.4). The cells were treated with the indicated concentrations of PI-88 or PM5 for 30 minutes before the addition of Waymouth’s containing 5% FBS. The cells were allowed to grow for 3 days before trypsinization, resuspension in Isoton II, and quantitation in an automated Coulter counter with coincidence correction.
ERK1/2 Phosphorylation Assay, FGF-2 ELISA, and Western Blot Analysis
Growth-arrested smooth muscle cells were incubated with PI-88 or PM5 for 30 minutes before injury by scraping.15 ERK1/2 activity was determined using the Amersham Biotrak p42/p44 kinase enzyme assay system in which a highly specific synthetic peptide substrate is phosphorylated (PLS/TP motif) with 32P by endogenous cellular ERK1/2. This assay is more specific than the commonly used substrate, myelin basic protein (MBP).
FGF-2 levels were determined in culture supernatant before and after incubation of the cells with PI-88, using a commercial enzyme-linked immunosorbent assay specific for this growth factor (R&D Systems).
Levels of FGF-2 and phospho-ERK protein in rat aortae (uninjured and 8 minutes after balloon injury), whose protein was extracted using TRIzol reagent (Invitrogen), were determined by Western blot analysis with antipeptide FGF-2 and phospho-ERK antibodies (Santa Cruz Biotechnology) and standard protocols.15
Blood Coagulation
Whole blood was collected by cardiac puncture into tubes containing 3.8% sodium citrate at a ratio of 5 parts blood to 1 part sodium citrate. Clotting times were determined using an automated coagulometer (Diagnostica Stago) standardized for tissue factor procoagulant activity.
Surface Plasmon Resonance
Binding studies were performed on a BIAcore 2000 surface plasmon resonance-based biosensor (BIACORE AB) using bovine lung heparin immobilized to a CM5 sensor chip (BIACORE). During immobilization, the flow rate of the running buffer (PBS buffer, pH 7.2, containing 10 mmol/L EDTA and 0.05% Tween 20) was maintained at 5 µL/min and the temperature at 25°C. The carboxymethylated surface of the sensor chip was first activated with 35 µL of a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-diethylaminopropyl) carbodiimide (EDC) (0.05 mol/L NHS, 0.2 mol/L EDC in distilled H2O), then 35 µL of NeutrAvidin (100 µg/mL in 10 mmol/L sodium acetate pH 4.5; Pierce) was injected over the activated surface for covalent attachment. Initial experiments indicated that FGF-2 yielded a high nonspecific binding response to the blank sensor chip. This is presumably due to the electrostatic interaction between the cationic growth factor and the negatively charged carboxymethyl dextran layer on the chip surface. By using NeutrAvidin instead of streptavidin, we were able to eliminate this nonspecific binding problem. Residual activated ester groups were blocked by the injection of 35 µL of 1 mol/L ethanolamine hydrochloride pH 8.5, followed by washing with 10 µL of 10 mmol/L HCl, to remove noncovalently bound material. Ten microliters of 4 mol/L NaCl was injected over the surfaces, and then nonspecific binding of FGF-2 (0.3 µmol/L in running buffer) to both the uncoupled and NeutrAvidin-coupled flow cells was measured and found to be insignificant on the NeutrAvidin-coupled flow cells. Fifteen microliters of biotinylated bovine lung heparin (1 µg/mL in 10 mmol/L sodium acetate pH 4.5) was then injected into the NeutrAvidin-coupled flow cell. The bovine lung heparin (Sigma) was biotinylated with a 25 molar excess of NHS-biotin (Pierce). Finally, flow cells were washed with 40 µL of 4 mol/L NaCl. For Kd determination, FGF-2 was diluted in running buffer to give a series of concentrations ranging from 0.002 µmol/L to 0.35 µmol/L. The flow rate was adjusted to 40 µL/min. Each FGF-2 concentration (325 µL of each) was injected over all 4 flow cells. Simultaneous measurements were thus obtained from one flow cell containing NeutrAvidin (background binding) and from the other flow cell containing biotinylated heparin on NeutrAvidin (specific binding). Data points were collected continuously during the binding and dissociation phases, and analysis was based on the response once saturation binding had been reached. Between each cycle, the derivatized sensor surface was regenerated with 40 µL of 4 mol/L NaCl. The sensorgrams were prepared for analysis with the aid of the BIAevaluation software version 3.0.2 (BIACORE AB). Baselines were adjusted to zero for all curves and background sensorgrams subtracted from experimental sensorgrams to produce curves of specific binding. The curves were then fit to determine the response at equilibrium for each concentration of FGF-2. The Kd value determined, is the weighted mean value from several such experiments in which the data were plotted as double-reciprocal plots and the intercept on the x-axis taken as -1/Kd. For inhibition binding studies, the effect of PI-88 (0, 0.008, 0.016, and 0.024 µmol/L) on the binding of FGF-2 (0.016, 0.02, 0.026,0.04, and 0.08 µmol/L) to the immobilized heparin was tested. Saturation binding curves for each of the FGF-2/PI-88 concentrations were obtained and the response at equilibrium determined, as described above. Double-reciprocal plots of response versus FGF-2 concentration, for each particular PI-88 concentration, were plotted. A primary plot was then obtained by plotting slope versus PI-88 concentration and the true inhibitory constant (Ki) determined from the point where the line intersected the x-axis. The experiment was done three times, and the Ki value given is the weighted mean of the three Ki values. When the reproducibility of the biosensor’s response to FGF-2 at a given concentration (0.025 µmol/L) was tested, the mean response was 350.5 and the standard deviation, 17.8. PI-88 was also tested alone in the concentration range being used and found not to give a response on its own.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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PI-88 Inhibits Vascular Smooth Muscle Cell Proliferation
The sulfated oligosaccharide PI-88 has previously been reported to inhibit tumorigenesis, metastasis, and angiogenesis in animal models.1 However, PI-88 has not yet been tested for its ability to influence the proliferative response to mitogenic stimuli in any cell type. Vascular smooth muscle cell proliferation and accumulation is a key feature of intimal thickening in the balloon-injured artery wall.10,16 We determined the effect of PI-88 on the proliferative response of growth-quiescent primary rat aortic smooth muscle cells to serum. PI-88 inhibited smooth muscle cell growth in a dose-dependent manner (Figure 1). In contrast, PM5, a minimally-sulfated oligosaccharide and backbone counterpart of PI-88,1 failed to influence smooth muscle cell proliferation even at concentrations as high as 200 µmol/L (Figure 1).
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PI-88 Inhibits Intimal Thickening After Balloon Angioplasty of Rat and Rabbit Carotid Arteries
Intraarterial balloon angioplasty to the rat common carotid artery leads to neointima formation and a reduction in luminal diameter within 14 days. Neointima formation, expressed as a percentage of the total cross-sectional area of the artery17,18 was 57.7±3.2% in rats in which saline was administered subcutaneously (Figure 2). In contrast, subcutaneous administration of PI-88 at a dose rate of 35 mg/kg per day, which commenced immediately after arterial injury and maintained for 14 days, significantly reduced (P=0.0008) the extent of intimal thickening (36.0±3.5%) (Figure 2). This dose of PI-88 was based on the amount of drug that inhibits tumor growth and metastasis in rats.1 Histological examination of the luminal surface of the dilated arteries showed a complete absence of an endothelial cell lining (Figure 2). The neointima was composed mainly of smooth muscle cells and amorphous extracellular matrix (ECM) (Figure 2).
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The rabbit model was used to illustrate the capacity of PI-88 to inhibit postangioplasty restenosis in a second and larger animal model, the rabbit.19 Neointimal thickening after 14 days occupied a cross-sectional area of 25.2±3.3% (Figure 3) or a neointima/media ratio of 0.37 (Table 1). The two lower doses of PI-88 (0.15 and 1.5 mg/kg per day) did not affect the area of neointima, but the higher dose (15 mg/kg per day) significantly reduced (P=0.003) intimal thickening (12.0±0.6%) (Figure 3). PI-88 (15 mg/kg per day) caused a marked reduction in intimal area (to 0.14) without any significant change in medial area (Table 1).
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PI-88 Blocks Heparan Sulfate Degradation After Balloon Injury of Rabbit Carotid Arteries
We have previously shown in culture that heparan sulfate maintains smooth muscle cells in the "contractile," nonproliferative phenotype, and that its pericellular removal is a trigger for phenotypic change and responsiveness to mitogens.20,21 We have also shown a profound reduction in both heparan sulfate and chondroitin sulfate within 6 hours of balloon injury in rabbit arteries preceding change in smooth muscle cell phenotype and formation of a neointimal thickening.22 Based on the capacity of PI-88 to inhibit heparanase activity in vitro,1 we hypothesized that heparanase blockade by PI-88 after balloon injury would result in decreased heparan sulfate proteoglycan (HSPG) degradation in the artery wall.
We analyzed histological sections of rabbit carotid artery, both uninjured and 4, 7, and 14 days after balloon catheter injury, for the distribution of heparan sulfate, chondroitin sulfate, and smooth muscle -actin in both the presence and absence of PI-88 administration. PI-88 (15 mg/kg per day) selectively blocked the loss of heparan sulfate without affecting chondroitin sulfate levels (data not shown), consistent with PI-88 inhibition of heparanase in vitro (Figure 4).
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PI-88 also blocked the change in the smooth muscle phenotype as measured by ultrastructural morphometric analysis of volume fraction of myofilaments (Vvmyo). PI-88 had no effect on the Vvmyo in smooth muscle cells 4 days after injury (48.4±2.3% in controls versus 46.3±12.0% in PI-88 treated). However, 7 days after injury, when the Vvmyo in untreated injured arteries had decreased markedly (28.9±0.5%), this effect was abrogated by PI-88 (Vvmyo 47.2±2.2, P=0.028). These data are supported by our previous demonstration that addition of exogenous arterial heparan sulfate to balloon-injured arteries inhibits the change in smooth muscle phenotype after injury.19
PI-88 Blocks MAP Kinase Activation but Augments FGF-2 Release by Vascular Smooth Muscle Cells After Injury
ERK1/2 is known to play a positive regulatory role in smooth muscle cell proliferation and intimal thickening after balloon angioplasty because ERK1/2 blockade inhibits smooth muscle cell hyperplasia.23 Based on our demonstration that PI-88 inhibits smooth muscle cell proliferation (Figure 1) and neointima formation in rat and rabbit arteries (Figures 2 and 3), we hypothesized that PI-88 may influence ERK1/2 activity in smooth muscle cells after injury. PI-88 (25 µmol/L) blocked ERK1/2-specific phosphorylation 8 minutes after injury, with no further inhibition at 100 µmol/L (Figure 5A). In contrast, the same concentration of PM5 had no inhibitory effect (Figure 5A). ERK1/2 activation within minutes of injury is due, at least in part, to the release and paracrine activity of endogenous FGF-2.15 Incubation of cultured smooth muscle cells with PI-88 (25 and 100 µmol/L) elevated levels of immunoreactive FGF-2 in the supernatant 2-fold, whereas PM5 had no effect (Figure 5B). Injury produced a dramatic (18-fold) increase in FGF-2 levels within 2 minutes (Figures 5B and 5C, compare y-axes). This elevation was augmented 2-fold further by PI-88 (25 or 100 µmol/L) (Figure 5C) but was unaffected by PM5 (Figure 5C). These findings indicate that PI-88 stimulates rapid FGF-2 release from smooth muscle cells, potentiates growth factor release after injury, and inhibits ERK1/2 activation.
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Western blot analysis demonstrates FGF-2 loss from the rat artery wall within 8 minutes of injury (Figure 5D); this was inhibited by PI-88 (Figure 5D). Although it is possible that some FGF-2 might be sequestered in the matrix or at the cell surface, our results are consistent with other reports showing the local release of FGF-2 into the circulation within minutes of injury.4 PI-88 also blocked injury-inducible ERK phosphorylation after this time, without altering blood coagulation in these animals (Table 2). The overall anticoagulant activity of PI-88, incidentally, is approximately 6-fold less than that of heparin,3 with PI-88 having negligible anticoagulant activity via antithrombin III and exerting its anticoagulant effect predominantly via heparin cofactor II.
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PI-88 Competitively Inhibits the Binding of FGF-2 to Immobilized Heparin
Because FGFs bind sulfated sugars such as heparan sulfate on the cell surface and in the ECM, and our present data demonstrate that PI-88 inhibits ERK1/2 activity despite increasing FGF-2 levels, we hypothesized that PI-88 directly binds FGF-2, preventing its interaction with heparan sulfate proteoglycan (HSPG). Determination of a true Ki value for inhibition of FGF-2 binding to immobilized heparin by PI-88 first requires determination of the Kd for FGF-2 binding to heparin. We performed this analysis using an optical biosensor that measures interactions directly in real time. Analysis of the double-reciprocal plot (1/response versus 1/FGF-2 concentration, Figures 6A and 6B) gave a Kd value of 7.9±0.5 nmol/L (Figures 6A and 6B). This is in good agreement with values previously determined by others that generally range between 1 and 10 nmol/L, using affinity electrophoresis,24 fluorospectrometric analysis,25 and evanescent wave biosensor analysis.26 We next examined the capacity of PI-88 to inhibit the binding of FGF-2 to the immobilized heparin. A more direct measure of the interaction between PI-88 and FGF-2 could not be performed because PI-88 cannot be biotinylated and hence immobilized on the surface of the chip, nor does it elicit a response when added as the analyte. Figure 6B illustrates a double-reciprocal plot for the system in the presence and absence of PI-88. The inhibition was found to be linear competitive because a replot of slope versus PI-88 concentration produced a straight line (data not shown). The fact that PI-88 is a competitive inhibitor indicates that it competes with heparin for the same binding site on FGF-2. The true Ki, the concentration of PI-88 required to inhibit 50% of FGF-2 binding to heparin, was determined to be 10.3±1.0 nmol/L. The failure of PM5 to inhibit the growth factor from binding to heparin, further suggests that a specific binding interaction is occurring between FGF-2 and PI-88 (data not shown). Thus, PI-88 acts as a competitive inhibitor of FGF-2 binding to cell surface and matrix HSPG.
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Mode of Action of PI-88
Balloon angioplasty is a widely used procedure to revascularize patients with occlusive coronary disease. The success of angioplasty, however, is hampered by restenosis, or renarrowing of the vessel wall, which occurs within a few months of the procedure.27 Restenosis, the consequence of smooth muscle cell accumulation in the intima,28 has long been the focus of interventional strategies to maintain patency long term. Insights from animal models have revealed that balloon injury stimulates medial smooth muscle cell proliferation and migration within a few days,29 with neointimal thickening maximal by 14 days.10,30 In this study, we evaluated the capacity of a recently developed polysulfated oligosaccharide to inhibit this process of arterial repair. Pump-based subcutaneous or intraperitoneal administration of PI-88, initiated immediately after balloon angioplasty of the carotid artery, significantly inhibited neointima formation in two separate animal models.
The actual mechanism of PI-88 inhibition has not previously been addressed, although comparison with PM5 indicates that sulfation is required for its biological activity.1 The present study clearly shows that two distinct mechanisms likely account for PI-88 inhibition of intimal thickening: first, inhibition of heparanase and, second, displacement/blockade of FGF-2 bioactivity (Figure 7). Heparan sulfate proteoglycans (HSPGs), found in the extracellular matrix and at the cell surface, maintain smooth muscle cells in the "contractile" nonproliferative phenotype.19,20,21,31 Heparanase activity is increased in the artery wall after injury.32 Inhibition of heparanase would limit matrix HSPG degradation by heparanases derived from invading leukocytes and platelets, thereby limiting the change in smooth muscle phenotype.20,21 In the present study, we show that PI-88, a potent inhibitor of in vitro tumor cell heparanase, did prevent the loss of its substrate heparan sulfate, suggesting this mechanism is active in the artery wall. PI-88 may, in addition, inhibit heparanase degradation of the vascular basement membrane, thereby preventing smooth muscle migration from the media into the intima or indeed the infiltration of circulating progenitor cells because these cells constitute up to 50% of neointimal cells after severe vascular injury.33 Alternatively, PI-88 binds to and prevents growth factor binding to cell surface HSPG,1,34 which serve as low-affinity cell-surface receptors for growth factors such as FGF-2.35 Previously, we showed that FGF-2 is released within minutes of cellular injury in vitro and activates ERK1/2 in a paracrine manner.4,15 FGF-2 is released in rat carotid arteries after balloon injury.36,37 It is also released in human coronary arteries on stenting.4 The low molecular weight heparan sulfate mimetic may release HSPG-bound FGF-2 from the cell-surface and extracellular matrix, inhibiting proliferation by disrupting the FGF-2/FGFR interaction. The nonsulfated version of PI-88 (PM5) is inactive as the sulfate groups of heparan sulfate are essential for FGF-2 binding. Thus, our present findings suggest that PI-88 blocks intimal thickening by its capacity to inhibit heparanase activity and/or sequester growth factor thereby preventing early signaling induced by injury. It is unclear which of these two mechanisms plays a more critical role in PI-88’s inhibition of intimal thickening.
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Acknowledgments |
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This study was funded by Progen Industries Limited, Brisbane, Australia, a NSW Department of Health Research and Development Infrastructure Grant, and a Program Grant from the National Health and Medical Research Council of Australia. L.M.K. is a Principal Research Fellow and J.H.C. is a Senior Principal Research Fellow of the NHMRC. We wish to thank Dr Craig Freeman for his assistance in preparing the manuscript, Professor John Morrison for analysis of the biosensor data, Dr Sue Brown for assistance in operating the biosensor, Tina Chua for the electron microscopy sections, and Charlotte Wheeler for proofreading.
Received December 12, 2002; revision received March 5, 2003; accepted April 1, 2003.
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