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Circulation. 1995;91:2972-2981

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(Circulation. 1995;91:2972-2981.)
© 1995 American Heart Association, Inc.

Articles

Failure of Heparin to Inhibit Intimal Hyperplasia in Injured Baboon Arteries

The Role of Heparin-Sensitive and -Insensitive Pathways in the Stimulation of Smooth Muscle Cell Migration and Proliferation

Randolph L. Geary, MD; Noriyuki Koyama, PhD; Trevina W. Wang, BS; Selina Vergel, BA; Alexander W. Clowes, MD

From the Department of Surgery and the Regional Primate Research Center at the University of Washington School of Medicine, Seattle, and the Department of Surgery of the Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC (R.L.G.).

Correspondence to Alexander W. Clowes, MD, Department of Surgery, BB420, PO Box 356410, University of Washington School of Medicine, Seattle, WA 98195-6410.


*    Abstract
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 *Abstract
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 down arrowMethods
down arrowResults
down arrowDiscussion
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Background Heparin is a potent inhibitor of smooth muscle cell (SMC) growth and intimal hyperplasia in animal models but has been ineffective in inhibiting restenosis in humans. This difference may relate to flaws in clinical study design or, alternatively, to interspecies differences in SMC response to heparin. To determine whether heparin could inhibit intimal hyperplasia in a species more closely related to humans, we studied the effect of a low-molecular-weight heparin (LMWH) on baboon SMC proliferation and migration in culture and in arteries subjected to experimental angioplasty.

Methods and Results LMWH or saline was infused continuously after experimental angioplasty of baboon peripheral arteries (six animals per group). After 28 days, bromodeoxyuridine (BrdU) was given to label proliferating cells, and balloon-injured arteries were perfusion-fixed in situ and removed for analysis. All arteries had reendothelialized (Evans blue dye exclusion). LMWH increased partial thromboplastin time (LMWH, 81.7±8.4 seconds versus saline, 34.7±0.8 seconds [mean±SEM]; P=.004) but failed to inhibit intimal thickening or SMC proliferation (intimal area: LMWH, 0.19±0.03 mm2 versus saline, 0.21±0.03 mm2; BrdU labeling: LMWH, 2.9±0.6% versus saline, 2.4±0.4%; P=NS). In culture, LMWH and standard heparin (100 µg/mL) significantly inhibited serum-induced but not platelet-derived growth factor (PDGF-BB)–induced SMC proliferation (% control, serum: LMWH, 60.5±4.0%, P=.0002; standard heparin, 29.4±8.2%, P=.0001; % control, PDGF-BB: LMWH, 117.7±11.3%, P=NS; standard heparin, 90.9±14.4%, P=NS) and SMC migration (% control, serum: LMWH, 15.3±1.9%, P=.0198; standard heparin, 26.4±13.8%, P=.0032; % control, PDGF-BB: LMWH, 98.5±14.3%, P=NS; standard heparin, 100.0±13.5%, P=NS).

Conclusions LMWH failed to inhibit intimal hyperplasia in a baboon angioplasty model. Furthermore, LMWH blocked serum-induced but not PDGF-BB–induced SMC proliferation and migration in culture. Thus, heparin-sensitive and -insensitive pathways exist for SMC activation. The relative importance of each pathway induced by injury may vary between species and thus account for different responses to heparin.


Key Words: heparin • angioplasty • restenosis • growth substances • muscle, smooth


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
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Current methods of correcting symptomatic arterial stenoses are mechanical and unavoidably injure treated arterial segments. Regardless of the method of reconstruction, 20% to 50% of treated arteries reaccumulate an excess amount of intimal tissue at the site of repair.1 2 3 4 5 This intimal "hyperplasia" is considered an exaggeration of normal healing mechanisms and leads to significant morbidity from recurrent luminal stenosis. Vascular smooth muscle cell (SMC) growth is central to the pathobiology of injury-induced intimal hyperplasia and restenosis,6 7 and strategies to improve the durability of vascular reconstructions have appropriately targeted SMC proliferation and migration.1 3

Heparin is a potential pharmacological inhibitor of intimal hyperplasia after vascular injury because of its dual anticoagulant and antiproliferative effects. Heparin inhibits SMC growth in vitro and in vivo.8 9 10 11 12 13 14 15 In the injured rat carotid artery, heparin decreases intimal thickening by inhibiting SMC proliferation and migration and by altering the extracellular matrix composition in the forming neointima.12 13 16 Heparin also suppresses the expression of matrix-degrading proteases that may be crucial for SMC movement and replication.17 18 19 20 Biochemical signaling pathways that mediate the effects of heparin are only now being elucidated.21 22 23

No pharmacological agent has consistently inhibited intimal hyperplasia in humans and improved long-term patency of arterial reconstructions.24 25 26 27 28 29 To date, results of using heparin as an inhibitor of intimal thickening are contradictory. Heparin inhibits baboon, human, bovine, and rat SMC proliferation in culture.17 30 31 32 In the rat carotid balloon injury model, heparin blocks SMC proliferation and intimal thickening.8 33 These results suggest that heparin should be effective at inhibiting restenosis in humans, but the clinical trials of heparin in coronary balloon angioplasty have been negative.27 28 34 35 36 There are many possible explanations for this discrepancy, including inadequate trial design and species differences in susceptibility to the drug. This concept is supported by studies of other pharmacological agents effective in animal models but not in humans, such as the angiotensin-converting enzyme (ACE) inhibitors. ACE inhibitors reduce experimental intimal hyperplasia in the rat37 but have been ineffective in human restenosis trials.38 39 This negative clinical result is mirrored in data from nonhuman primate studies demonstrating that ACE inhibitors fail to decrease intimal thickening in grafts and arteries subjected to angioplasty and endarterectomy.40 It is possible, then, that rodent and other nonprimate models of arterial injury might not predict the response to pharmacological inhibitors in humans, but more closely related primate species might. Before accepting the lack of inhibition in humans, we studied the effect of heparin in a baboon model of experimental angioplasty. If intimal hyperplasia is the major factor contributing to restenosis and if baboons and humans are similar in regard to vascular healing, then heparin might not inhibit intimal hyperplasia in balloon-injured baboon arteries.

Our experiments were designed to determine whether low-molecular-weight heparin (LMWH) could block SMC growth in vivo under a regimen that has worked in rats. Since we found that, at the dose used, LMWH did not block intimal hyperplasia, we sought an explanation to resolve the differences in response of baboon SMCs to heparin in vivo and in vitro. We suggest that there may be heparin-sensitive and heparin-insensitive pathways for activation of SMC migration and proliferation. The importance of specific pathways induced by arterial injury may vary among species and therefore determine the response to heparin.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
down arrowDiscussion
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Reagents
LMWH (SR 80258A, Sanofi Recherche) was prepared by periodate degradation of injectable-grade standard heparin followed by ß-elimination under basic conditions, reduction in sodium borohydride, and ethanol fractionation.15 SR 80258A has an average molecular weight of 6000 D, an antithrombin activity of 6 IU/mg, an anti–factor Xa activity of 2 IU/mg, and an anticoagulant activity (activated partial thromboplastin time, aPTT) of 5 IU/mg. Plasma levels of LMWH were measured by Dr Jean-Claude Lormeau of Sanofi Recherche. In brief, 96-well polystyrene plates were precoated with poly-L-lysine and washed with PBS. Plasma samples were added and incubated for 10 minutes at room temperature. After washing with PBS, a solution of LMWH covalently conjugated to peroxidase was added to each well and incubated for 10 minutes at room temperature. Plates were again washed, bound LMWH reacted with hydrogen peroxide/o-phenylene diamine, and the reaction product was quantified spectrophotometrically by its absorbance at 492 nm. Plasma concentrations were interpreted from a standard curve constructed from plates preincubated with plasma containing known quantities of LMWH. Proliferation and migration assays were repeated using both LMWH and standard commercial heparin (Sigma Chemical Co). Recombinant human platelet-derived growth factor (PDGF)-BB was provided by Dr Charles E. Hart of Zymogenetics Inc.

Animal Model
Balloon catheter denudation of saphenous arteries was performed in 12 male baboons (Papio cynocephalus) weighing {approx}10 kg. Anesthesia was induced with ketamine hydrochloride (10 mg/kg IM) and maintained with inhaled halothane. Cefazolin sodium (25 mg/kg, Bristol-Myers Squibb) was administered intramuscularly, and both femoral arteries were exposed from midthigh to the bifurcation into saphenous and popliteal arteries at the knee. All animals received a dose of standard heparin (100 U/kg IV, Elkins-Sinn, Inc) immediately before balloon denudation. The common femoral artery was occluded, and a 3F balloon arterial embolectomy catheter (V. Mueller Inc) was inserted through a side branch 1 to 2 cm above the bifurcation and passed 4 cm into the saphenous artery. The balloon was then inflated and retrieved under tension while the shaft of the catheter was twisted. After three passes, the catheter was removed and flow restored. For uniformity, the same individual performed all balloon injuries. Before surgery, animals were selected randomly for intravenous infusion of either LMWH (0.6 mg · kg-1 · h-1 in sterile saline) or saline by means of four mini osmotic pumps (Alzet 2-ml-4, Alza Corp). This dose of LMWH was determined in a pilot study to be the maximal dose that could be given without hazardous anticoagulation. At the time of balloon denudation, preprimed pumps were placed into the retroperitoneum and connected to Silastic tubing inserted into the vena cava. This provided immediate drug delivery (or saline) after balloon denudation. Wounds were infiltrated with bupivacaine hydrochloride (Marcaine 0.25%, Winthrop Pharmaceuticals), and the animals were returned to single-animal cages until they had fully recovered from surgery. Animals received oral acetaminophen (Children's Tylenol, 80 mg/6 h, McNeil) for 2 days after surgery.

Animals were examined, and blood was drawn to measure coagulation parameters (complete blood count, platelet count, aPTT, prothrombin time, thrombin time, and fibrinogen) under ketamine sedation (10 mg/kg IM) immediately before arterial injury and at 1, 7, 14, 21, and 28 days thereafter. Plasma levels of LMWH were measured (see above) at 7 and 28 days after injury. After 28 days, animals were euthanatized, and the vasculature was fixed by perfusion in situ. Animals received 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU, 30 mg/kg in saline [30 mg/mL], Boehringer Mannheim Inc) 1, 9, and 17 hours before death. Standard heparin (300 U/kg IV) and Evans blue dye (50 mg/kg, 50 mg/mL in 0.9% NaCl IV) were administered and were followed by a barbiturate overdose. The arteries were then perfused under pressure (100 mm Hg) via the aortic arch with lactated Ringer's solution (3 L) and then with 10% neutral buffered formalin (3 L). The femoral and saphenous arteries were removed en bloc and placed into fresh formalin for 48 hours before paraffin embedding. Osmotic pumps were removed, and the residual volume of fluid was measured to calculate actual drug delivery to each animal.

All animal care and procedures were performed at the University of Washington Regional Primate Research Center in accordance with state and federal laws. Animal protocols were approved by the University of Washington Animal Care Committee and conformed to guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and by the National Institutes of Health publication No. 86-23, Guide for the Care and Use of Laboratory Animals.

Morphology
Fixed saphenous arteries were cut into segments 5 mm long for paraffin embedding. Sections 5 µm thick were cut from each block and stained with Verhoeff's–van Gieson's stain for morphometric analysis. Cross sections were projected onto a computerized digitizing pad with a microscope and camera lucida, and the luminal, intimal, and medial areas of each cross section were measured. Mean values for each injured artery were determined by averaging cross-sectional measurements from the four central adjacent rings of injured artery segments.

Immunocytochemistry
To determine cellular composition and proliferation (see below) in the intima and media, paraffin-embedded sections from ballooned arteries were deparaffinized in xylene, rehydrated in graded alcohols, and immunostained. For cell type identification, antibodies specific to SMC {alpha}-actin (Boehringer Mannheim Inc), endothelial cell factor VIII–related antigen (DAKO Corp), and macrophage CD-68 antigen (DAKO) were applied. Primary antibodies were localized with appropriate biotinylated secondary antibodies and tertiary avidin-biotin-peroxidase staining (Vector Laboratories Inc). Control slides were included, with appropriate nonimmune IgG used as the primary antibody. Sections were counterstained and examined by standard light microscopy.

Cell Proliferation After Injury
Animals received intramuscular BrdU at 1, 9, and 17 hours before death. Deparaffinized 5-µm sections from each graft ring were stained with a monoclonal antibody against BrdU (Boehringer Mannheim) as described above. After being counterstained with hematoxylin, slides were examined under oil with a x100 objective. Proliferating intimal and medial SMCs were identified by dark brown nuclear staining. Total intimal and medial SMC numbers were estimated for each section by multiplying the intimal or medial cross-sectional areas (see above) by the number of nuclei per square millimeter (estimated by counting nuclei in eight representative high-power fields [HPF] of defined area with an eyepiece reticle). The BrdU labeling index (%) in the intima or media was calculated by dividing the number of labeled SMCs by the total number of SMCs and multiplying by 100. A mean labeling index for each artery was determined by averaging values from the same four rings of each artery as used for morphometry (see above).

SMC Culture and In Vitro Proliferation and Migration Assays
To determine whether heparin inhibition of SMC proliferation varied depending on the mitogen used, cultures were established from the aorta, internal jugular vein, and saphenous arteries of three individual baboons by the explant method as previously described.41 For proliferation assays, cells were studied between passages 3 and 6 and plated at 2.0x104/cm2 in Dulbecco-Vogt medium containing 5% fetal bovine serum (FBS; GIBCO) and 0.1% MITO+ serum extender (Collaborative Research). After 24 hours, the cells were growth-arrested for 3 days in serum-free medium (Dulbecco's modified Eagle's medium [DMEM]/Ham's F-12 [1:1] containing 6 µg insulin/mL, 5 µg transferrin/mL, 1 mg ovalbumin/mL, 200 U penicillin/mL, and 200 µg streptomycin/mL).42 Experiments were started by the addition of fresh medium containing 10% FBS or PDGF-BB (10 ng/mL) with or without heparin (100 µg/mL LMWH or standard heparin). Controls received a change of serum-free medium. After 24 hours, [3H]thymidine (1 µCi/mL, New England Nuclear) was added for 4 hours. Cells were rinsed once with PBS and then washed three times with cold trichloroacetic acid (TCA) (1 mL, 5% solution). The TCA-precipitable fraction was solubilized with 0.25 mol/L NaOH. The amount of [3H]thymidine incorporated was determined by scintillation counting of aliquots of solubilized TCA-precipitated DNA. Experiments were performed in duplicate or triplicate. Inhibition of SMC proliferation by heparin was calculated as follows: Inhibition (% control) = (cpm mitogen+heparin/cpm mitogen alone), where cpm is counts per minute of [3H]thymidine-containing TCA-precipitated DNA, mitogen is either serum or PDGF-BB, and heparin is either LMWH or standard heparin. To document that heparin did not affect [3H]thymidine transport, autoradiography was performed in one experiment in parallel with DNA extraction for [3H]thymidine uptake. For autoradiograms, plates were fixed in methanol, dipped in Kodak NBT-2 emulsion, and stored at 4°C for 7 days. Plates were then developed and stained with hematoxylin, and the number of labeled nuclei (those with at least five overlying silver grains) was determined by counting 500 cells per well with triplicate wells per treatment condition. The labeling index was calculated by dividing the number of labeled nuclei by the total number of nuclei counted and multiplying by 100.

SMC migration was assayed by a modification of the Boyden chamber method with 48-well microchemotaxis chambers (Neuro Probe Inc) and polycarbonate filters (Nucleopore Corp) with pores 10 µm in diameter.43 Filters were precoated with 2.7 µL/well of basement membrane solution (Matrigel, Collaborative Research Inc) and were allowed to dry for storage. Before use, Matrigel-coated filters were reconstituted with 10 µL distilled water at 37°C for 30 minutes. Cultured baboon aortic SMCs were trypsinized and suspended at a concentration of 5.0x105 cells/mL in serum-free DMEM. Forty microliters of the SMC suspension was added to the upper chamber, and 25 µL of DMEM with either 2% FBS (vol/vol) or 10 ng/mL PDGF-BB with or without heparin (100 µg/mL of either LMWH or standard heparin) was added to the lower chamber. The chamber was then incubated at 37°C in 5% CO2 in air for 6 hours. The filter was removed, and the SMCs on the upper side were scraped off. SMCs that had migrated through the Matrigel layer to the lower side of the filter were fixed in methanol, stained with Diff-Quick, and counted with an x100 objective to determine migrating cell number per HPF. Migration activity was expressed as the average number of cells that had migrated through the Matrigel per HPF. Migration activity in controls (serum or PDGF-BB alone) was set to 100% and the activity in heparin groups expressed as percent of control. Migration assays were repeated four times and performed in triplicate.

Statistical Analysis
Comparisons were made between injured arteries in LMWH and saline treatment groups by nonparametric analysis (Mann-Whitney U test). Variables compared include luminal, intimal, and medial areas, intimal and medial BrdU labeling indexes, aPTT, hematocrit (Hct), and plasma LMWH levels. For in vitro proliferation and migration assays, paired two-tailed Student's t tests were used to compare the heparin inhibition of serum-induced or PDGF-BB–induced SMC responses. Statistical significance was assumed for P<.05. Statistics were performed with STATVIEW-II software for the Macintosh (Abacus Concepts, Inc). Results are expressed as the mean±SEM and n=6 unless otherwise stated.


*    Results
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*Results
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Baboon Model of Intimal Hyperplasia
All animals remained healthy and without evidence of bleeding complications throughout the period of study. Infusion rates were the same for LMWH and saline treatment groups (delivery: 10.9±0.1 µL/h LMWH versus 10.5±0.2 µL/h saline, P=NS). Subsequently, the treated group received 0.6±0.01 mg · kg-1 · h-1 LMWH. Plasma LMWH levels were significantly elevated at both 7 and 28 days after injury in treated animals (Table 1Down). High plasma levels of LMWH achieved in treated animals resulted in a significant increase in aPTT. Values were increased within 1 day of injury and remained elevated throughout the period of study (Table 1Down). While the mean Hct was lower in the LMWH group, the change from preoperative to ending Hct (28 days) was not significantly different in the two study groups (change in Hct: LMWH, -1.5±2.4% versus saline, 1.3±1.2%, P=.37). Other hematologic and coagulation parameters (platelet count, prothrombin time, thrombin time, and fibrinogen) were normal in both treatment groups (data not shown). Of the 12 saphenous arteries injured in each treatment group (2 per animal), 11 arteries in the saline group and 8 in the LMWH group were patent at the end of the study. In animals with 2 patent arteries, morphometric values and labeling indexes in each were averaged to determine a mean value for each animal. In those animals with single patent arteries, measurements from that vessel were used for data analysis.


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  		Table 1. Coagulation Parameters After Experimental Angioplasty

Lesions in injured arteries were similar in the two treatment groups (Fig 1Down). Uninjured baboon saphenous arteries contained few SMCs beneath the endothelium (no developed intimal layer) overlying the internal elastic lamina. Intimal thickening was not present in uninjured arteries, whereas concentric lesions were present in injured arterial segments (Fig 2ADown and 2BDown). Injured segments were uniformly reendothelialized in the two treatment groups at 28 days as shown by Evans blue dye exclusion (data not shown). Complete endothelialization was confirmed by immunostaining for factor VIII–related antigen (Fig 2CDown). The newly formed intima was populated primarily by SMCs (Fig 2DDown). Many macrophages were seen in the adventitia (not shown) but only rarely in the media or intima of injured arteries (Fig 2EDown).



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  		Figure 1. Photomicrographs of injured artery cross sections. Lesions in arteries from low-molecular-weight heparin–treated (A) and saline-treated (B) baboons were similar in appearance 28 days after experimental angioplasty (original magnification x250).



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  		Figure 2. Photomicrographs of low-molecular-weight heparin–treated injured artery cross sections. Cross-sectional analysis of baboon arteries 28 days after experimental balloon angioplasty revealed no apparent differences with either low-molecular-weight heparin or saline treatment. Uninjured regions in baboon saphenous arteries (A, Verhoeff's–van Gieson's stain) demonstrate a muscular artery without a preexisting intima. Twenty-eight days after experimental angioplasty (B, Verhoeff's–van Gieson's stain), a circumferential intimal lesion has formed that on closer examination (C) is covered by an intact endothelial monolayer, as demonstrated by factor VIII–related antigen staining. Both the intima and media are composed predominantly of smooth muscle cells expressing {alpha}-actin (D). Macrophages, stained for CD-68 antigen (arrow), were seen frequently in the adventitia (not shown) but rarely in intimal lesions or in the media (E). BrdU staining localized many proliferating cells within the intima, often in the region nearest the lumen (F) (original magnification: A and B x63; C, D, E, and F x250).

The average cross-sectional areas of the lumen, intima, and media were similar in injured arteries in LMWH or saline treatment groups. Likewise, the intima-to-media ratio was not altered by LMWH treatment (Table 2Down). SMC proliferation was low in the media (LMWH, 0.6±0.3% versus saline, 0.5±0.2%; P=NS) and relatively high in the intima (LMWH, 2.9±0.6% versus saline, 2.4±0.4%; P=NS) in both treatment groups (Table 2Down) compared with quiescent levels in the media of uninjured control vessels (<0.1%).


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  		Table 2. Morphometry and Smooth Muscle Cell Proliferation

Effects of Heparin on Baboon SMC Proliferation and Migration In Vitro
To further assess the effects of heparin on SMC growth, proliferation, and migration, assays were performed in culture. LMWH inhibited serum-induced proliferation in SMCs derived from saphenous arteries, the aorta, and jugular vein similarly but failed to inhibit PDGF-BB–induced proliferation (Table 3Down). In a representative experiment, LMWH inhibited serum-induced SMC proliferation by 42%, whereas PDGF-BB–induced proliferation was inhibited by only 13% (SMC thymidine incorporation [cpm]: serum-free, 1015 cpm; 10% serum alone, 9576 cpm; serum plus LMWH, 5541 cpm; PDGF-BB, 34 756 cpm; PDGF-BB plus LMWH, 30 397 cpm). Similarly, LMWH inhibited serum-induced migration by baboon aortic SMCs in a Boyden chamber assay but failed to inhibit PDGF-BB–induced migration (SMC migration [cells per HPF]: serum alone, 77±9; serum plus LMWH, 12±1; PDGF-BB alone, 89±9; PDGF-BB plus LMWH, 88±13; n=3 experiments). Results were similar for experiments using standard heparin in both proliferation and migration assays (data summarized in Tables 3Down and 4Down).


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  		Table 3. Heparin Inhibition of SMC Proliferation in Culture


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  		Table 4. Heparin Inhibition of SMC Migration in Culture


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
In the present study, a continuous intravenous infusion of LMWH failed to inhibit intimal hyperplasia in a nonhuman primate model of angioplasty. This lack of inhibition contrasts with experimental data from our group and others demonstrating heparin inhibition of SMC growth in culture and of intimal hyperplasia in other animal models of arterial injury.8 9 10 11 12 13 14 15 On the other hand, the present results are consistent with negative clinical trials of heparin in the prevention of restenosis after coronary angioplasty.27 28 34 35 36 A number of factors may be contributing to differences in heparin effect among experimental and clinical models of restenosis. To be considered are variations in the heparin preparation; differences in the route, dose, and duration of heparin administration; differences in the mechanisms of arterial injury and subsequent lesion formation; and interspecies differences in SMC growth control. We present evidence that heparin-sensitive and heparin-insensitive pathways exist for the stimulation of baboon SMC proliferation and migration in culture. Both LMWH and standard heparin inhibit proliferation and migration induced by serum but not by PDGF-BB. The specific growth factors stimulating SMC proliferation and migration in vivo probably vary between species. The heparin sensitivity of the predominant mitogens may well determine the degree of heparin inhibition for a given species.

In the present study, it is unlikely that the heparin preparation or dosage accounts for the lack of inhibition. The LMWH preparation used in the baboon injury model has previously been shown to inhibit serum-induced baboon SMC proliferation in culture.17 In addition, this preparation of LMWH has effectively inhibited SMC proliferation and intimal thickening after balloon injury in the rat carotid artery.18 The dose used in the present study (0.6 mg · kg-1 · h-1) is slightly less than the amount (1.0 mg · kg-1 · h-1) that reduced intimal hyperplasia by 50% in the rat carotid model.18 Despite a low anti–thrombin III activity (6 IU/mg), this high dose of LMWH doubled the aPTT in treated baboons within 24 hours of injury, and this level of anticoagulation was maintained throughout the period of study. Plasma LMWH levels measured during the period of study confirmed drug delivery (17.2±6.9 and 10.4±2.7 µg/mL at 7 and 28 days, respectively). In the rat study,18 plasma LMWH levels were in the range of 5 to 15 µg/mL as measured with the same assay (AW Clowes and YPT Au, unpublished results, 1992). Taken together, these data demonstrate that the heparin preparation and dose used in the present study were equivalent to those effective at inhibiting lesion formation in the rat.

On the other hand, a recent preliminary report of a similar study in baboons found inhibition of intimal thickening with a similar modified heparin (Astenose, Glycomed Corp).44 The distinct difference between these two studies is the much higher dose administered in the Astenose study. Plasma concentrations of Astenose were more than fourfold greater (65.0±8.6 µg/mL at 30 days) than in the present study, and this resulted in significant systemic anticoagulation (aPTT, 197 seconds at 30 days versus 37 seconds at baseline) (Dr Josiah N. Wilcox, personal communication, December 14, 1994). Interestingly, Astenose had no effect on SMC proliferation in the media or intima at 7 and 28 days, respectively, suggesting an effect primarily on SMC migration. Perhaps the LMWH used in the present study would inhibit lesion formation at much higher doses, but this, in our view, would carry an inordinate risk of bleeding complications.

Previous work by this group and others suggests that the timing and route of administration are crucial in obtaining inhibition of intimal hyperplasia after experimental angioplasty.12 16 45 46 47 In the rat carotid injury model, heparin must be started within 24 to 48 hours of injury16 and continued for 4 to 7 days12 45 to inhibit intimal thickening. In the rat, heparin is most effective if given as a continuous intravenous or periarterial infusion rather than by intermittent subcutaneous dosing as used in human clinical protocols.35 47 In fact, the intermittent subcutaneous route has caused paradoxical thrombosis46 and exacerbation of intimal thickening.47 Based on these results, we administered LMWH as a continuous intravenous infusion via implanted pumps. An intravenous bolus of standard heparin was given immediately before the experimental angioplasty, and the LMWH infusion was begun during the procedure and continued throughout the 28-day study.

Intimal Hyperplasia as the Paradigm for Restenosis
The best-characterized experimental model of human intimal hyperplasia is the ballooned rat carotid artery, and comparisons with other models of arterial reconstruction may help to explain variations in heparin effects. Ballooning of the rat carotid artery removes the endothelium and damages the underlying media, causing a loss of 20% to 30% of medial SMCs. Platelets immediately carpet the denuded luminal surface. Despite the degree of injury, thrombus formation and arterial occlusion are uncommon. Within 24 to 36 hours of injury, a population of medial SMCs begins to proliferate, and by 4 days, proliferating and quiescent medial SMCs begin to migrate into the forming intima. SMCs replicate further in the intima and produce extracellular matrix. Beyond 2 weeks, intimal SMC proliferation subsides, and further thickening is due to accumulation of extracellular matrix. Endothelial regrowth is limited to only a few millimeters. Denuded regions longer than 3 cm fail to heal and are covered by a layer of SMCs. In the rat model, heparin inhibits the initial wave of medial SMC proliferation, the migration of SMCs from media into intima, SMC proliferation within the intima, and extracellular matrix accumulation.

The present study in baboons was modeled after the rat carotid balloon-injury experiments. The baboon saphenous artery was selected because it has few branches near its origin, which limits the sources of endothelial ingrowth. The location of the injury is unlikely to explain the outcome. Heparin has been effective at inhibiting SMC growth in vivo in a number of animal models at sites other than the carotid artery. For instance, heparin inhibits intimal proliferation in stented pig coronary arteries,48 in rat and rabbit arteriovenous grafts,49 50 and in rabbit aorta and iliac arteries after angioplasty.51 In the present study, SMCs from different anatomic sites responded similarly to both LMWH and standard heparin in proliferation assays in culture (Table 3Up). This result is in agreement with a recent report demonstrating consistent responses to heparin in human SMCs isolated from peripheral arteries or saphenous veins from individual patients.52

Experimental angioplasty was performed as in the rat but with an appropriate-size catheter (3F). Baboon lesions resembled those in the rat in that they tended to be circumferential and slightly eccentric in thickness and generally failed to produce stenoses. As in the rat, the intima is composed predominantly of SMCs surrounded by extracellular matrix with few macrophages at 28 days.

An intriguing difference between the rat and baboon is the presence of a regenerated endothelial cell monolayer in all injured baboon arteries. In contrast to the rat, in which intimal thickening is inhibited beneath regenerated endothelium, baboon lesions developed despite the endothelium. Endothelial cells produce endogenous inhibitors of SMC growth, such as heparan sulfate proteoglycans, which could decrease lesion growth and thereby diminish the apparent inhibitory effects of heparin. Alternatively, endothelium may alter the bioavailability of heparin to underlying SMCs.

Experimental models of restenosis assume that SMC proliferation and intimal hyperplasia are central to lesion formation in humans. In fact, injury to normal arteries, as in the rat, may not be representative of the more complex lesions formed in angioplastied human atherosclerotic arteries. Because human lesions are generally not available, they cannot be analyzed in the same manner as experimental animal tissues. The pathogenesis of human restenosis is therefore largely inferred. Recent analysis of human lesions obtained from failed vascular grafts and from coronary atherectomy specimens has cast doubt on paradigms set forth by animal models of intimal hyperplasia. Rekhter and colleagues53 reported very high proliferative rates (18% to 24%) in anastomotic lesions from failed arteriovenous grafts, but the proliferating cells were associated primarily with microvessels rather than the neointima. A contrasting report by O'Brien and colleagues54 failed to show significant SMC proliferation in a large number of atherectomy specimens removed at various times after coronary angioplasty. In regard to restenosis after angioplasty, intimal hyperplasia may well be a small component of a complex process involving vessel fracture, plaque compression, spasm, recoil, thrombosis, and remodeling.55 It may be unreasonable to expect heparin alone to impact such a heterogeneous process.

Biochemical Mediators of the Response to Heparin: Heparin-Sensitive and -Insensitive Pathways
The complex biochemical control of SMC migration and proliferation after injury and the modulation of these signals by heparin are poorly defined. Heparin has been shown to alter the binding of specific growth factors such as PDGF32 and to inhibit epidermal growth factor receptor expression.56 Heparin potentiates the inhibitory effects of transforming growth factor-ß by releasing it from a carrier protein57 and also inhibits second messenger pathways leading to key proto-oncogene expression important for cell cycling.21 22 Heparin selectively inhibits production of specific proteases important for degrading extracellular matrix, a critical process for cell movement and proliferation. These include tissue-type plasminogen activator, interstitial collagenase, stromelysin, and 92-kD gelatinase.17 18 19 20

Two examples of growth factors involved in the repair of arterial injury are basic fibroblast growth factor (bFGF) and PDGF. The initial wave of medial SMC proliferation in the rat is driven in part by bFGF released from injured or disrupted cells in the vessel wall.58 Blocking antibodies to bFGF decrease medial SMC proliferation by 80% to 90%.59 PDGF is a weak mitogen in the rat model but stimulates migration of SMCs into the intima.60 Blocking antibodies to PDGF inhibit the lesion area without affecting SMC proliferation.61 In the rat, heparin acts in part by displacing bFGF from the vessel wall after injury.62 Although heparin also reduces SMC migration, it is unclear whether this is due to interactions of heparin and PDGF or other factors such as matrix metalloproteases.

PDGF may be relatively more important in the primate response to arterial injury. Balloon injury in primates results in adherent platelet thrombus with platelet release of PDGF-BB (the predominant isoform in baboon and human platelets) onto the denuded artery wall. In addition, PDGF is expressed in human and primate atherosclerotic lesions, by vessel wall cells themselves, and by SMCs isolated from the intima of failed human arterial bypass grafts.7 63 64 PDGF is a potent mitogen for primate SMCs in culture. The PDGF-A gene is induced at the time of SMC proliferation in the intima of baboon vascular grafts in response to reduced shear stress.65 Our results in the present study suggest that PDGF-induced SMC proliferation and migration in vitro are insensitive to heparin inhibition. If PDGF or other heparin-insensitive factors are important in the primate response to arterial injury, then heparin may not be effective at inhibiting lesion formation.

The concept of heparin-sensitive and -insensitive pathways for SMC activation is supported by work reported by Wright et al66 and by Ottlinger et al.21 In these studies, heparin inhibited expression of the proto-oncogenes c-fos and c-myc in response to phorbol ester or serum stimulation but not in response to epidermal growth factor stimulation. Subsequent experiments have shown that proto-oncogene activation is mediated through mitogen-activated protein kinase activation, which is in turn inhibited by heparin when stimulated by phorbol myristate acetate or serum but not when stimulated by epidermal growth factor. PDGF stimulation also leads to mitogen-activated protein kinase activation, and it will be important to determine whether this signaling in baboon SMCs is resistant to heparin inhibition and whether phorbol myristate acetate- or serum-stimulated pathways are sensitive to heparin.

In summary, LMWH given in high doses appears to be ineffective at inhibiting SMC proliferation and intimal hyperplasia in a baboon model of angioplasty. This lack of effect is in agreement with trials of heparin in the prevention of coronary restenosis in humans. We propose that the lack of a LMWH effect in baboons might reflect the presence of a heparin-insensitive pathway of SMC activation (possibly via PDGF). A test of this hypothesis will require in vivo experiments with specific inhibitors (eg, anti-PDGF antibodies).


*    Acknowledgments
 
This study was supported in part by grants HL-30946 and RR-00166 from the National Institutes of Health. The authors would like to thank Dr Jean-Claude Lormeau (Sanofi Recherche–Centre Choay, Paris) for the generous gift of the SR 80258A heparin and for performing plasma SR 80258A assays and Zymogenetics, Inc, Seattle, for the generous gift of the recombinant human PDGF-BB.

Received October 27, 1994; revision received December 19, 1994; accepted December 27, 1994.


*    References
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