Low Molecular Weight Fucoidan Prevents Neointimal Hyperplasia in Rabbit Iliac Artery In-Stent Restenosis Model

Intimal hyperplasia due to migration and proliferation of smooth muscle cells (SMCs) from the media to the intima is a major component of restenosis after stent implantation.^1,2 Polysaccharides constitute a large family of molecules capable of developing molecular interactions with cellular targets within the arterial wall.³ For instance, heparin, a natural sulfated polysaccharide, displays pleiotropic effects independent of the anticoagulant activity, including SMC growth inhibition,⁴ anti-inflammatory activity,⁵ and growth factor protection.⁶

Fucoidan is a sulfated polysaccharide extracted from brown seaweed that reduces rat SMC proliferation in vitro in a more intensive manner than heparin.⁷ We recently succeeded in producing and characterizing new homogeneous fractions of low molecular weight (LMW) fucoidan with low anticoagulant activity.

The aims of the present study were to investigate the ability of LMW fucoidan to regulate vascular SMCs. For this purpose, we first tested the ability of LMW fucoidan to inhibit rabbit SMC proliferation in vitro. We explored the pharmacokinetics of LMW fucoidan injected in rats and the effect of LMW fucoidan on intimal hyperplasia. The results described below indicate that LMW fucoidan is a strong inhibitor of intimal hyperplasia and may be potentially relevant for the treatment of in-stent restenosis.

Methods

Polysaccharides

LMW fucoidan was isolated and hydrolyzed by a radical depolymerization process⁸ from high molecular weight (HMW) extracts of brown marine algae. The characteristics of LMW fucoidan according to previously reported analytical methods⁹ are as follows: weight-average molecular mass 8±1 kDa; fucose content 35% (wt/wt); uronic acid content 3% (wt/wt); and sulfate content 34% (wt/wt). The anticoagulant activity in vitro of the LMW fucoidan was measured by an activated partial thromboplastin time (APTT), and the amount of LMW fucoidan required to obtain an APTT of 80 seconds (control 40 seconds) was 25 μg/mL.¹⁰ The anticoagulant activity in vivo of LMW fucoidan was measured in rabbits by APTT and prothrombin time after intramuscular injection of 5 mg/kg LMW fucoidan. LMW fucoidan was fluorolabeled with the use of 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein.^7,11 LMW heparin (6±2 kDa) was commercially available from Sigma Chemical Co.

Cell Culture

Eagle’s minimum essential medium (MEM) and trypsin-EDTA were purchased from GIBCO. Calf serum was obtained from Eurobio. SMCs were isolated from the abdominal aorta of male New Zealand White rabbits.¹² Cells were cultured at 37°C in a humidified atmosphere (5% CO₂) in MEM supplemented with 10% serum and 2 mmol/L l-glutamine. Third-passage cultures were used throughout the present study.

Growth Studies

Rabbit SMCs (1.2×10⁴) in MEM containing 10% serum were growth-arrested by placing them in medium with 0.4% serum for 72 hours. Cell growth was followed in MEM+10% serum with or without polysaccharides. Toxicity was evaluated by trypan blue and clonal growth assay as described.⁷ Thymidine uptake at 24 hours was assessed in quadruplicate wells with 1 μCi per well of 6-[³H]thymidine that was pulse-labeled for 60 minutes. Cells were washed, DNA was precipitated with 10% trichloroacetic acid, and radioactivity was counted. Inhibition of SMC proliferation was assessed at day 3 by cell counting of serum-stimulated SMCs in the absence or presence of LMW fucoidan or LMW heparin.

Confocal Study

SMCs plated at low density on coverslips were incubated at 37°C with fluorescent LMW fucoidan (5 μg/mL) for the indicated times, washed 4 times with PBS containing 0.2% BSA, and fixed in 3.7% formaldehyde solution.⁷ Cultured SMCs were observed under a confocal microscope (Leica SP; excitation wavelength [λex] 488 nm, emission wavelength [λem] 500 to 590 nm).

Pharmacokinetic Experiments

Animal protocols were approved by the Institutional Animal Care and Use Committee of Faculté Bichat. Adult male Wistar rats (280 to 300 g) were anesthetized with intraperitoneal pentobarbital (0.1 mL/kg). A silicon-tipped polyethylene catheter was inserted into the right jugular vein. The bladder was exteriorized and catheterized. A bolus of 5 mg/kg fluorescent LMW fucoidan was injected (1) intravenously via the jugular vein catheter followed by saline solution to flush the catheter or (2) intramuscularly in the right leg. Blood samples were obtained from the catheter at 0, 15, and 30 minutes and at 1, 2, 4, and 6 hours. Urine was collected via the bladder catheter. Blood was centrifuged, and the serum was obtained. The concentration of fluorescent LMW fucoidan was measured in the plasma and urine by a spectrofluorometer at λex and λem of 489 and 515 nm, respectively.

Pharmacokinetic Analysis

Equation 1 describes the time course of fluorescent LMW fucoidan concentration in the blood according to a 2-compartment open model: equation

where A and B are constants. The parameters were determined using the nonlinear least-squares program Micropharm.¹³

The elimination half-time period, t_1/2β (β phase), corresponds to the time taken for the concentration of drug in plasma to decline to half of its original value and was calculated from the parameter β by Equation 2: equation

The clearance (Cl) is the elimination of the drug in milliliters per minute expressed by Equation 3: equation

where AUC is the area under the curve and was calculated by using the trapezoidal rule by the Micropharm program, and D is the administered dose of LMW fucoidan. The apparent distribution volume is the ratio between the amount of drug in a whole organism and its blood or plasma concentration measured at the same time. Percentage of urinary excretion was calculated from the administered dose and the urinary excreted dose.

Angioplasty and Stenting Protocol

Male New Zealand White rabbits (3.5 to 4 kg) received a standard diet. Animals were anesthetized with intravenous pentobarbital, and the right carotid artery was catheterized with a 5F sheath. A 3.0-mm-diameter 20-mm-long angioplasty balloon catheter was advanced over a standard 0.014-in flexible guidewire in both iliac arteries. The iliac arteries were injured by 3 successive 1-minute inflations at 10 atm with a 1-minute reperfusion phase after each inflation. In some experiments, 1 mL fluorescent LMW fucoidan (5 mg/mL) was locally delivered with a 3-mm annular balloon catheter (Nycomed) in the rabbit iliac artery after angioplasty. After 5 minutes, a segment of damaged iliac artery and an adjacent segment of artery were excised, washed with PBS, and observed under a fluorescent microscope. For the stented animals, a 15-mm-long metallic stent (Helistent-Hexacath, Reuil Malmaison) mounted over the balloon was implanted in both iliac arteries immediately after balloon angioplasty (30-second inflation at 10 atm).¹⁴ LMW fucoidan at 5 mg/kg was injected intramuscularly twice a day for 14 days. Five animals (2 stents per animal) were treated with LMW fucoidan for 14 days. Five control animals received saline.

Tissue Harvest and Histology Processing

Fourteen days after stenting, rabbits were killed by pentobarbital overdose. Iliac arteries were perfusion-fixed and impregnated in methacrylate as described.¹⁴ Four-micron arterial sections were cut with tungsten carbide knives and stained with hematoxylin-eosin, Masson’s trichrome, or orcein. In each artery, 3 sections were taken: 2 at 2 mm from the distal ends of the stent and 1 in the middle. Cell density in the neointima was observed under a microscope and analyzed (Lucia Software, Nikon) for 10 different sections per group.

Morphometric Analysis

Digital planimetry with the use of a video camera mounted on a microscope analyzed the borders of the external elastic lamina, internal elastic lamina, and vessel lumen. A computer program allowed quantification of the intimal, medial, and luminal areas.¹⁴ Intimal growth was estimated by using the intimal area, the luminal area, the ratio of intimal to medial areas, and the ratio of the intimal area to the area bounded by the internal elastic lamina (luminal cross-sectional area narrowing).

Statistical Analysis

Results are expressed as mean±SD. The significance of the differences between groups was analyzed by 1-way ANOVA followed by post hoc tests. Values of P<0.05 were considered significant.

Results

LMW Fucoidan Inhibits Rabbit SMC Growth In Vitro

We tested the ability of LMW fucoidan to inhibit rabbit SMC growth in vitro. No cell toxicity was observed even at the highest dose (1000 μg/mL). [³H]Thymidine uptake indicated that SMC DNA synthesis 24 hours after serum stimulation was significantly (P<0.001) inhibited in the presence of LMW fucoidan in a dose-dependent fashion: 28±2%, 52±3%, and 78±2% inhibition at 10, 100, and 1000 μg/mL, respectively. LMW heparin was less potent on the inhibition of DNA synthesis with 21±2%, 31±4%, and 49±3% inhibition at 10, 100, and 1000 μg/mL, respectively. By cell counting at day 3, we found that LMW fucoidan was a better SMC growth inhibitor than LMW heparin (Figure 1).

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LMW Fucoidan Is Internalized in Cultured Rabbit SMCs

Because the growth-inhibitory effect of LMW fucoidan could be, at least in part, due to its internalization in SMCs, we first investigated its uptake by SMCs in vitro. The fate of LMW fucoidan added to cultured rabbit SMCs at 37°C was studied by confocal microscopy using 5 μg/mL of fluorescent LMW fucoidan. After incubation of SMCs, LMW fucoidan was internalized by endocytosis in fluorescent vesicles clearly seen at 6 hours (Figure 2A). The number of fluorescent vesicles in the perinuclear region increased at 24 hours, but nuclear internalization was never observed (Figure 2B).

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LMW Fucoidan Is Found on Injured Segments of Arteries After Balloon Angioplasty

We then investigated whether LMW fucoidan has an affinity for the arterial wall in vivo. A local delivery catheter was used to deliver fluorescent LMW fucoidan in a rabbit iliac artery after angioplasty. By fluorescence microscopy, an intense fluorescence was revealed on the segment damaged by angioplasty, whereas low fluorescence intensity was detected on the adjacent uninjured segment of the artery (data not shown).

A 2-Compartment Pharmacokinetic Model Describes LMW Fucoidan Distribution In Vivo

The serum concentrations of LMW fucoidan after intravenous or intramuscular injection in rats are displayed in Figure 3A and 3B, respectively. After intravenous injection, LMW fucoidan exhibited a biphasic decline with a rapid decrease of plasma concentration 2 hours after injection and a secondary slow linear decrease (Figure 3A). The pharmacokinetic parameters on 8 adults Wistar rats were as follows: clearance 0.88±0.46 mL/min, t_1/2β 56.5±25.0 minutes, and distribution volume 57.6±27.9 mL. Urinary excretion of LMW fucoidan was 33.2±2.1%. After intramuscular injection, plasma concentration of LMW fucoidan remained in the 10-μg/mL range for at least 6 hours (Figure 3B). Pharmacokinetic analysis revealed that a 2-compartment model described best the plasma behavior of LMW fucoidan.

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LMW Fucoidan Prevents In-Stent Intimal Hyperplasia in Rabbit Iliac Arteries

No rabbit died during the procedures or the 14-day treatment with LMW fucoidan (5 mg/kg IM). Prothrombin time and APTT were not different in blood samples obtained from LMW fucoidan–treated animals versus control animals (data not shown).

In the animal model, angioplasty (3 successive inflations at 10 atm) and stent implantation for 14 days in rabbit iliac arteries increased (P<0.0001) the intimal area of these arteries (1.79±0.43 mm²) compared with arteries in uninjured animals (0.013±0.002 mm²), whereas the medial area was not different (0.48±0.06 mm² in uninjured group versus 0.39±0.09 mm² in stented group).

After angioplasty and stent implantation, LMW fucoidan treatment after 14 days (Figure 4) was associated with a thinner (P<0.001) fibrocellular neointima (101±20 μm) compared with control stenting (236±90 μm). Cell density in the intima was not different (P=0.5) between the 2 groups of stented animals, but the significant decrease of the size of the intima by fucoidan treatment suggested an important limitation of the total number of invading and proliferating SMCs. Morphometric analyses indicated that arterial cross-sectional area was similar in the 2 groups, indicating that stent-induced arterial injury was of similar magnitude in the 2 groups without adverse effect of LMW fucoidan on media integrity (Table). Interestingly, intimal area with LMW fucoidan was significantly (P<0.0001) reduced by 59±11%, the intima/media ratio was reduced by 56±12%, and luminal cross-sectional area narrowing was reduced by 58±10% (Table). In turn, the luminal area of arteries from LMW fucoidan–treated animals was increased (Table, Figure 4).

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Discussion

The results described above indicate that LMW fucoidan is a strong inhibitor of intimal hyperplasia in vivo and may be potentially relevant in the treatment of in-stent restenosis. Fucoidans are a family of l-fucose–containing sulfated polysaccharides extracted from brown seaweed, Ascophyllum nodosum.⁸ This vegetal polymer shares chemical analogies with heparin, a sulfated polysaccharide of animal origin, but exhibits lower anticoagulant activity.⁷

Continuous administration of HMW fucoidan (>100 kDa) has been shown to reduce intimal hyperplasia after balloon injury in rats.¹⁵ Nevertheless, the requirement for continuous administration limits the use of HMW fucoidan for pharmaceutical and clinical applications. In addition, restenosis was induced in the study of McCaffrey et al¹⁵ with angioplasty alone, ie, without stent implantation. Surprisingly, there are no published data on the inhibitory effect of LMW fucoidan on intimal hyperplasia. Thus, we decided to test the efficacy of LMW fucoidan on intimal growth after stenting in the rabbit, a model that is more relevant to modern clinical restenosis than is balloon abrasion of the rat carotid artery. The data presented indicated that LMW fucoidan reduced neointimal hyperplasia by ≈60% and increased the luminal area by ≈25%, making this compound one of the most efficient ever tested in experimental restenosis by using a noncontinuous administration regimen.¹⁶ In contrast to heparin tested in animal models of balloon angioplasty^17,18 and clinical trials,¹⁹ HMW fucoidan was already used at high dose (25 mg/kg in mice,²⁰ 100 mg/kg in rats,²¹ and 10 mg/kg per hour in rabbits²²) without bleeding. In the present study, the fraction of LMW fucoidan (8 kDa), used in rabbits at low dose (5 mg/kg), exhibited no anticoagulant activity in vivo.

In agreement with previous studies using HMW fucoidan in cultured rat and human SMCs,^7,23,24 LMW fucoidan reduced rabbit SMC proliferation in vitro and appeared to be a more antiproliferative agent than LMW heparin. Our confocal microscopy studies show that LMW fucoidan has high affinity for cultured SMCs and is internalized in perinuclear endocytotic vesicles. In a previous study using a 20-kDa fucoidan fraction,^7,25 we demonstrated that the antiproliferative effect of fucoidan on rat SMCs was mediated by its binding to membrane sites, probably similar to those that mediate the endocytosis of heparin.^12,26 Perinuclear localization of LMW fucoidan suggests an antiproliferative activity via intracellular signaling pathways.^23,24 We also observed that after angioplasty and local injection of LMW fucoidan in rabbit iliac arteries, the polysaccharide was located on the injured segment of the artery, whereas a low binding was noticed on the artery without angioplasty. This preferential binding in vivo after injury by angioplasty strongly suggested a favorable location of the LMW fucoidan to inhibit SMC activation and proliferation. McCaffrey et al²⁷ suggested that antiproliferative activity of HMW fucoidan on rat and bovine aortic cultured SMCs was linked to the protection of transforming growth factor-β1 from proteolytic degradation by plasmin and trypsin. We have previously observed an action of HMW fucoidan on growth factors, such as fibroblast growth factor-1 and fibroblast growth factor-2, leading to an increase in endothelial cell proliferation in vitro²⁸ as well the release of tissue factor pathway inhibitor from cultured human endothelial cells,²⁹ which may be relevant in vivo for LMW fucoidan. In vitro and in vivo signaling pathways for the inhibition of SMC growth and migration by LMW fucoidan remain to be fully characterized.

The antiproliferative effect on SMCs of LMW fucoidan probably does not fully explain its potent inhibitory effect on intimal hyperplasia. At least 4 other features of fucoidan may play a role as well: (1) The 2-compartment model that best described the plasma behavior of LMW fucoidan after intravenous administration suggests a rapid organ or cellular uptake followed by a slow decrease. Heparin is cleared through a combination of a rapid mechanism (via endothelial cells and macrophage) and much slower first-order mechanisms via renal excretion.³⁰ Prolonged high plasma rates of LMW fucoidan after intramuscular administration may also facilitate molecular interactions with membrane receptors present on SMCs, which are in contact with blood after the destruction of the endothelial layer by angioplasty. (2) HMW fucoidan is known to modulate leukocyte activation and adhesion via interaction with selectins.³¹ Monocyte activation and adhesion are important stimuli in in-stent restenosis.³² It has been demonstrated that anti-inflammatory cytokines and monoclonal antibody targeted against monocyte integrins inhibit monocyte infiltration and reduce in-stent intimal hyperplasia.¹⁴ HMW fucoidan reduces in vivo neutrophil adhesion and leukocyte migration.^33,34 Our recent data indicate that sulfated polysaccharides, including fucoidan, inhibit the release of proinflammatory cytokines/chemokines by activated monocytes,³⁵ which may be relevant for the inhibition of intimal hyperplasia.³⁶ (3) We and other groups have described that HMW and LMW fucoidans have antithrombotic activities and reduce platelet aggregation, an important stimulus of restenosis.^10,29,37 (4) HMW fucoidan increases the mobilization of stem/progenitor cells³⁸ and increases plasma levels of stroma-derived factor 1, a highly potent chemoattractant for leukocytes and stem/progenitor cells.³⁹ Because circulating progenitor cells are involved in reendothelialization of injured arteries,⁴⁰ a mobilization by LMW fucoidan may result in reduced intimal growth.

In conclusion, we reported in the present study promising results of intimal hyperplasia inhibition with a LMW fucoidan in stented rabbit iliac arteries. The low anticoagulant activity, the favorable safety profile, the potent antiproliferative effect on SMCs, and the potential anti-inflammatory properties of LMW fucoidan make it a promising candidate for prevention of in-stent restenosis either systematically or locally via local delivery catheters or coated stents.^41,42

Footnotes