Induction of neointimal hyperplasia by coronary angioplasty balloon overinflation: Comparison of feeder pigs to Yucatan minipigs

Humphrey

17.

18.

19.

20.

21.

et al.

concentration) in patients without traditional evidence of acute myocardial infarction. A risk indicator of coronary death. Eur Heart J 1992;13:1387-92. The Committee on Enzymes of The Scandinavian Society for Clinical Chemistry and Clinical Physiology (SCE). Recommended method for the determination of creatine kinase in blood modified by the inclusion of EDTA. Stand J Clin Lab Invest 1979;39:1-5. Stein W, ed. Laboratory diagnosis of acute myocardial infarction. Using CK and CKMB immunoinhibition. Darmstadt, Germany: GIT Verlag 1988;30-62. H$rder M, Gerhardt W. Diagnostic application of biochemical markers in acute myocardial infarction. In: Thygesen K, Kjekshus J, eds. Myocardial ischaemia. Oxford: Blackwell Scientific Publications, 1990:251-9. Hunt AC, Chow SL, Shiu MF, Chilton DC, Cummins B, Cummins P. Release of creatine kinase-MB and cardiac specific troponin I following percutaneous transluminal coronary angioplasty. Eur Heart J 1991;12:690-4. Talasz H, Genser N, Mair J, Dworzak ER, Friedrich G, Moes

American

January 1990 Heart Journal

N, Miihlberger V, Puschendorf B. Side-branch occlusion during percutaneous transluminal coronary angioplasty. Lancet 1992;339:1380-2. 22. Cummins B, Cummins P. Cardiac specific troponin-I radioimmunoassay in the diagnosis of acute myocardial infarction.

AM HEARTJ1987;113:1333-44. 23. Katus HA, Remppis A, Scheffold T, Diederich KW, Kuebler W. Intracellular compartmentation of cardiac troponin T and its release kinetics in patients with reperfused and nonreperfused myocardial infarction. Am J Cardiol 1991;67:1360-7. 24. Bush HS, Ferguson JJ, Angelini P, Willerson JT. Twelve-lead electrocardiographic evaluation of ischaemia during percutaneous transluminal coronary angioplasty and its correlation with acute reocclusion. AM HEART J 1991;121:1591-9. 25. Droste C, Roskamm H. Ambulatory electrocardiography evaluation of the post-coronary artery bypass graft and post-percutaneous transluminal coronary angioplasty patient. Diagnostic and prognostic value. In: Crawford MH, Kennedy HL, eds. Cardiology clinics. Ambulatory electrocardiography. Philadelphia: WB Saunders, 1992:431-48.

Induction of neointimal hyperplasia by coronary angioplasty balloon overinflation: Comparison of feeder pigs to Yucatan minipigs We evaluated the use of simple balloon overinflation to induce neointimal hyperplasia in a porcine model of coronary artery restenosis. By using standard percutaneous transluminal coronary angioplasty techniques, left anterior descending (LAD) and/or left circumflex (LCX) coronary arteries of either juvenile feeder pigs or adult Yucatan minipigs were intentionally overinflated. Four weeks later, resultant neointimal hyperplastic responses (neointimalmedia area; NIIM) were quantitated morphometrically. At all ballooned sites neointimal hyperplasia occurred only when the internal elastic lamina (IEL) was ruptured; the degree of hyperplasia correlated directly with the injury index, that is, the percentage of IEL circumference that fractured (r = 0.74; n = 25; p < 0.05). Despite similar injury indexes in the LAD bed, there was a trend (p = 0.07; analysis of variance) toward greater NllM ratios in the Yucatan minipig versus the feeder pig group (1.14 + 0.21 vs 0.73 + 0.09, n = ‘//group). We found no such trend in the LCX bed, where the injury index (25.7% f 3.5%) was significantly greater than that of the LAD (18.2% t 1.2%, p < 0.05). If variations in balloon-induced vascular injury are accounted for, the technique of balloon overinflation of coronary arteries should prove useful in testing potential antirestenotic agents in either adult or juvenile pigs. (AM HEART J 1994;127:20-31.)

William R. Humphrey, BS, Carol A. Simmons, BS, Christopher F. Toombs, PhD, and Ronald J. Shebuski, PhD Kalamazoo, Mich.

From the Department of Cardiovascular Diseases Research,.Upjohn Lahoratories. Received for publication December 21, 1992; accepted May 3, 1993. Reprint requests: Ronald J. Shebuski, PhD, Cardiovascular Diseases Research, Upjohn Laboratories, 301 Henrietta St., Kalamazoo, MI 49001. Copyright @ 1994 by Mosby-Year Book, Inc. 0002-8703/94/$1.00 + .lO 4/l/60644

Percutaneous transluminal coronary angioplasty (PTCA) has become a popular method of treating patients with ischemic heart disease, with over 300,000 procedures performed annually in the United States.’ However, the efficacy of PTCA in restoring coronary arterial blood flow in the long term is lim-

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ited by a high incidence (25 % to 40%) of restenosis that occurs within 3 to 6 months of the initial procedure.2-3 Much research has been directed toward defining the biologic processes that mediate this phenomenon. Vascular recoil/vasospasm4* 5; platelet-meand smooth-muscle cell diated thrombosis5-7* proliferation, migration, and matrix deposition7-lo are responses involved in the complex restenotic process. In the search for an effective antirestenotic regimen, extrapolation of experimental findings to the clinical setting has been relatively disappointing.*ir I2 This poor correlation between preclinical and clinical efficacy is not surprising considering the complexity of factors affecting the restenotic process, the numerous factors affecting drug pharmacology and pharmacokinetics, and the inherent dissimilarities that can exist between these factors when an an-

imal model is compared with the actual human condition. A variety of animal species, including ratsg, lo rabbits,l”, i4 and pigs, i5, l6 have been used to establish experimental models of restenosis after balloon injury. Of these species, the pig has the dual advantage of being readily available and having marked similarities to humans in terms of arterial morphology17 and composition of restenotic lesions that form after vascular insult.16, 18slg One of the first models developed to examine the vascular response to balloon injury in pigs focused on overinflation of the common carotid artery.r5 To address concerns that inherent

differences in the composition

of peripheral

versus

coronary arteries may limit the clinical relevance of a carotid injury model, various laboratories are now

evaluating the restenotic process in the coronary vasculature of pigs. 16,I872o Repeated balloon overinflation18s2o or placement of oversized endoluminal stentsmgr8 are two demonstrated methods of inducing a myointimal hyperplastic response in porcine coronary arteries. Although reported to have better consistency and produce more extensive proliferative

lesions, the use of oversized endovascular

stents has

two major drawbacks. First, the severity of injury introduced by chronic stent placement and the overwhelming neointimal hyperplastic response it in-

duces may mask the true effectiveness of potential antirestenotic agents. l, l8 Second, because the current clinical use of stents is limited to those PTCA patients experiencing acute or threatened closure and does not involve intentional oversizing of the device 21 the clinical relevance of oversized stent models df coronary arterial restenosis is questionable. The technique of simple balloon overinflation as an

alternative

to stent models in inducing

porcine cor-

onary artery neointimal hyperplastic responses has

et al.

21

been introduced in prior studies”> 2o but has not been thoroughly explored as a possible means of testing potential antirestenotic agents. Therefore we sought to closely examine this technique and define its reliability in inducing neointimal lesion formation in porcine coronary arteries within 4 weeks of the initial balloon injury. Coronary arterial balloon overinflation experiments were performed on three different groups of pigs: juvenile feeder pigs, juvenile feeder pigs placed on a high-lipid diet, and sexually mature Yucatan minipigs. With these experiments, we have examined what effects modifying serum lipid profiles or varying the sexual maturity-growth potential of the animal have on the reproductibility and/or quality of the hyperplastic responses induced by balloon overinflation. Additionally, we evaluated the relation between variations in the extent of balloon-induced

vascular damage and the resultant size of the neointimal lesions that developed. Furthermore, preliminary immunostaining was performed to begin characterization of the specific cell type(s) that comprise

the neointimal METHODS Animals

lesions that form in this model.

and diet considerations. Male and female domesticfeeder pigsobtained from local breeders(5 weeksto 3 months old, Yorkshire-Hampshire-Landrace crosses) and Yucatan minipigs (4 to 6 months old; Charles River Laboratories, Wilmington, Mass.) were the two strains of pigs usedin this study. All procedureswere performed in compliance with the Animal Welfare Act (regulations, 9 CFR parts 1,2, and 3) and the Guide for the Care and Use of Laboratory Animals, (DHEW Publication (NIH) 85-23, 1985). The pigs were maintained on a chow consistingof 16% crude protein and supplementedwith the antibiotic lincomycin (200gm/ton; Upjohn Laboratories, Kalamazoo, Mich.). The Yucatan minipigswere fed a maintenancediet of 1.2 kg feed/pig/day (4.5% body weight) throughout the experimental protocol according to supplier instructions. Consequently the feeder pigs were also administered a daily food ration of 4.5% body weight/day (assuminga weight gain of roughly 1 kg/day) to maintain similar caloric intakes betweengroups.In a separategroup of feeder pigs, weanedfeeder piglets (5 to 6 weeksold) were given a highlipid diet 30 to 40 days before and for 7 days after balloon overinflation. The high-lipid diet, similar to that described by Gerrity et al.,s2consistedof 20A crude protein and was supplementedwith 1.5% cholesterol, 19.5% lard, and lincomycin (200 gm/ton) . Percutaneous transluminal coronary angioplasty. The pigswereinitially pretreated with verapamil hydrochloride (120mg p.o.), 12to 16 hoursbefore angioplasty to minimize angioplasty-related coronary vasospasm.ls,23General anesthesiawas establishedwith intramuscular injections of ketamine hydrochloride (25mg/kg) and midazolamhydrochloride (0.6 mg/kg); animals were placed in a dorsal recumbent position. An intermediate doseof atropine (0.5

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mg, intramuscularly) was given to limit mucous secretions. Anesthesia was maintained for the next 2 hours with supplemental ketamine (20 to 30 mg/kg/hr) and midazolam (0.3 to 0.4 mg/kg/hr) intramuscular injections. With this anesthetic regimen the pigs were able to breath oxygenated room air unassisted. For the intravenous delivery of drugs, a 21.gauge butterfly catheter was then inserted and secured into a marginal ear vein. After local administration of 2 % lidocaine to the ventral surface of the neck, a 10 to 12 cm midline incision was made to expose the left common carotid artery. An 8F introducer sheath was secured in the artery to provide catheter access into the left coronary ostium. The introducer port was attached to a pressure transducer for continuous recording of arterial blood pressure and heart rate on a polygraph (model ‘7D Grass Instruments, Quincy, Mass.), Before further catheterization procedures, the pigs were given sodium heparin (200 U/kg + 50 U/kg/hr, intravenous bolus; Upjohn) and aspirin (5.0 mg/kg intravenously). Under fluoroscopic guidance (GE Mobile Fluoricon Fluoroscope, General Electric, Milwaukee, Wis.), a 7F to 8F JR guide catheter was positioned in the left coronary ostium, and angiograms were performed on the left anterior descending (LAD) and left circumflex (LCX) coronary vascular beds. All angiographic procedures were performed with one-plane fluoroscopy and recorded on VHS video tape for repeated playback and analysis. Immediately before coronary arterial advancement of a PTCA catheter, a 50 mg intravenous dose of the antifibrillatory and antiarrhythmic agent bretylium tosylate was given and an intracoronary injection of nitroglycerin (50 to 150 pg) to alleviate post-PTCA vasospasm. 23 Control angiograms were then replayed for micrometer analysis of coronary artery diameters. In a series of acute experiments, angiographitally determined LAD coronary arterial diameters correlated directly with diameter measurements made on fixed and embedded histologic sections taken from the corresponding angiographic site (r = 0.94, p = 0.005, n = 6), indicating that the fluoroscopic images provided an accurate estimate of relative arterial diameters. After micrometer analysis an oversized (3.0 or 3.5 mm) PTCA catheter (SCIMED Life Systems, Inc.; Maple Grove, Minn.) was advanced into the LAD vascular bed to a position where the diameter of the inflated balloon catheter was between l.land 1.4-fold greater than that of the artery. Once positioned, the PTCA catheter was inflated twice with 100% Conray contrast media (Mallinckrodt Medical, St. Louis, MO.). For each balloon inflation the inflation pressure and duration were 8 atm and 20 set, respectively, with a 60 set interval allowed between inflations. The balloon overinflation procedure was then repeated in the LCX vascular bed in some but not all of the pigs by using the same guide catheter positioning. Although suitable for the performance of PTCA procedures, l6 the right coronary arterial bed was not used in this study so that the surgical procedure would not be prolonged. After balloon overinflation the PTCA catheter was withdrawn, post-PTCA angiography was performed to confirm

American

January 1994 Heart Journal

vessel patency, and a follow-up intracoronary injection of nitroglycerin (50 to 150 pg) delivered. The coronary guide catheter and introducer sheath were withdrawn, the carotid artery ligated, and the neck wound closed in layers by using interrupted sutures. Prophylactic intramuscular injections of penicillin (1 million U) and dihydrostreptomytin (1.25 gm) were given to the animal, which was allowed to recover in its own pen over the next 2 to 3 hours. The recovery period extended to the next 4 weeks. Throughout this recovery period the pigs received daily oral doses of aspirin (SO mg/day). Serum lipid profiles. Serum samples were collected at the time of PTCA and stored at -20’ C for later analysis of lipid profiles. For feeder pigs placed on a high-lipid diets, serum lipid profiles were also measured on samples taken before diet implementation and at death or 3 weeks after the high-lipid diet was discontinued. Total serum cholesterol and triglycerides were measured on a Coulter Dacos analyzer by using the respective assay kits (Coulter Electronics, Hialeah, Fla.). Total serum lipid concentration was measured by using a spectrophotometric assay specific for unsaturated compounds. 24 Serum high-density lipoprotein (HDL) cholesterol levels were determined on a Roche Cobra Mira S analyzer by using the HDL cholesterol separating reagent and cholesterol reagent assay kits (Roche Diagnostic Systems, Nutley, N.J.). Histopathology. Four weeks after the PTCA event the pigs were reanesthetized with ketamine/midazolam (intramuscularly) and positioned as described above. Supplemental anesthesia was maintained with a continuous intravenous drip of ketamine (15 to 20 mg/kg/hr) plus fentanyl citrate (0.1 to 0.2 mg/kg/hr) via an external jugular vein catheter. This supplemental anesthetic regimen, which effectively maintains a surgical plane of anesthesia for extended periods of time, depresses spontaneous respiration. Thus the pigs were intubated and respired mechanically with a positive-pressure pump (tidal volume = 20 ml of oxygen-enriched room air/kg). The respiratory rate was initially set at 20 inflationslmin and was adjusted as needed to maintain arterial PCO~and pH within normal ranges. A midline incision was made in the ventral neck region to position an 8F introducer sheath in the right common carotid artery. The pigs were heparinized as before (200 U/kg plus 50 U/kg/l-n intravenously) and angiographic visualization of the LAD and LCX coronary vascular beds performed. A 2 ml/kg bolus intravenous injection of Evans blue dye (1% weight/volume solution in saline solution) was administered via the jugular vein catheter. The dye was allowed to circulate for 1 hour before death via intravenous sodium pentobarbital (50 mg/kg). With this staining method, the site(s) of previous balloon-induced arterial damage were consistently demarcated by increased Evans blue uptake into the vascular wall. After the pigs were dead, a sternotomy was performed to excise the heart, which was then attached via the ascending aorta to a peristaltic perfusion apparatus. The heart was then retrogradely perfused at 100 mm Hg with 0.9% normal saline solution for

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23

Table I. Success rates in achieving LAD coronary arterial lesions in pigs 28 days after PTCA overinflation

IEL,

Cause

Group

Domestic feeder pigs Normal diet High-lipid diet Yucatanminipigs

Injury Index =

kLf

IEL, Proliferation

Index =

x

100

!‘!! area M area

Fig. 1. Morphometric procedures usedto quantitate severity of vascular damage (injury index) and extent of neointimal (NI) hyperplasia (proliferation index) induced by balloon overinflation of porcine coronary arteries. IEL, Internal elastic lamina; IELf, IEL fracture length; IEL,, IEL circumference; M, media.

15minutes, followed by a 30-minute perfusion with fixative (Omni-Fix II, An-Con Genetics, Melville, N.Y.). In some experiments in which oil red 0 staining for lipids wasperformed, the perfusion fixative waschangedto a 2% glutaraldehyde, 1% paraformaldehyde solution in 0.1 M sodium cacodylate (pH 7.25). Three sequential 10mm coronary arterial segments(two regions of altered Evans blue dye uptake, one region of proximal or distal uninvolved coronary artery) were dissectedfrom each balloon-injured site after perfusion fixation, immersed in Omni-Fix II for > 24 hours, and prepared for paraffin embedding.Six cross-sectionswere cut (each6 pm thick and 150 pm apart) from the approximate center of each tissue block, with three of the sections stained with hematoxylin and counterstained with eosinphloxine to demarcatecell types; the remaining three sections were stained with aldehyde fuschin and counterstained with Van Gieson’ssolution to demarcate the IEL. Gluteraldhyde/paraformaldehyde-fixed coronary artery segmentswere obtained from representative animalsin all three groups. These segmentswere sectioned with a cryostat at -18’ C and stained with oil red 0 to examine the extent of lipid deposition in the lesions. Morphometric measurementsof up to nine coronary arterial cross-sections(six sectionsfrom the 20 mm area of Evans blue dye uptake; three sectionsfrom normal region) were evaluated from eachballoonedregion by usinga computer-assistedimageanalysissystem(The Microscope Co., Medina, Ohio). Fig. 1 illustrates the method of morphologic assessmentof vascular injury based on severity of IEL rupture (injury index) and the corresponding extent of neointimal hyperplasia based on the neointimal/medial

Lesion frequency

7110 418 7110

Death

of failure IEL intact

2 3 2

L,.Q, Left anterior descending; PTCA, percutaneous transluminal angioplasty; IEL, internal elastic lamina. *Morphologic evaluation of cross-sections of ballooned LAD artery sections revealed no IEL rupture(s).

*

1 1

1 coronary coronary

area ratio (proliferation index). With this technique, estimation of the original IEL circumference (IEL,) at the ballooned site included the estimated length of IEL fracture. Although a potential sourceof error, the estimated IEL, calculated in this way correlated well with the intact IEL,s measuredfrom either proximal or distal nonballooned regions. The injury and proliferative indexes obtained from cross-sectionsof the sameballoon-injured site were averagedto yield one value/PTCA site. lmmunohistochemistry. To confirm the presence of smooth musclecells in neointimal lesionsthat formed at sites of balloon overinflation, immunohistochemistry was performed on representative LAD lesionsharvested from all three experimental groups. Paraffin sectionsof porcine LAD coronary artery were deparaffinized in limonenelxylene (3:1), followed with rinsesin 100% and 70% ethanol, and rehydrated in Tris buffer (pH 7.5) (Biomeda Corp., Foster City, Calif.). These sectionswere treated with 0.1% trypsin solution for 30 minutes at 37’ C to enhanceimmunoperoxidase staining. A monoclonal antibody specific againsta-smooth musclecell actinz5 (SigmaChemical Co., St. Louis, MO.) and a labeledstreptavidin biotin peroxidase kit (DAK0 Corp., Carpinteria, Calif.) were usedto stain for cr-smoothmuscle cell actin according to the kit protocol. Mouse myeloma protein of the sameimmunoglobulin Gza isotype and samedilution of the primary antibody served as a negative control. Statistical analysis. All values not representing frequency of occurrence were expressedas mean + SEM. Differencesbetweengroupsin achieving neointimal lesions were compared by using chi-square analysis. One-way analysis of variance (ANOVA) followed by Fisher’s least significant difference test were used to determine differencesbetween groupsregarding extent of balloon overinflation, coronary arterial proliferation and injury indexes, control hemodynamics,body weights, and serumlipid profiles. If Bartlett’s test indicated an inequality of variances between groups, values of the given parameter were logtransformed before ANOVA analysis. Linear regression analysis was used, when appropriate, to determine relationships between measuredparameters. Differences be-

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Fig. 2. Histologic sectionsof porcine LAD coronary artery harvested 4 weeksafter balloon overinflation. A, In regionswhere IEL furrow) was not disrupted, neointimal lesion formation wasnot observed. B, Eccentric neointimal (M) lesionsformed in regionswhere IEL wasruptured. (Original magnification X160.)

tween groups were considered statistically significant if p 50.05.

RESULTS Histopathology, mortality, and exclusions. Intentional balloon overinflation of porcine LAD coronary arteries induced neointimal hyperplasia within 4 weeks in 18 of 28 experiments. The frequency of hyperplasia was consistent between groups (Table I). Mortality and balloon inflations insufficient to cause IEL rupture were the two causes of failure in generating coronary arterial neointimal lesions. Of the seven animals that did not survive the procedure, causes of death included LAD dissection and immediate vascular collapse (n = 3), ventricular fibrillation (n = 3), and delayed acute myocardial infarction associated with platelet-mediated thrombosis (n = 1). Excluding those animals that did not survive the procedure, neointimal hyperplasia was obtained in 18 (>85%) of the 21 LAD coronary arteries that were ballooned. In all cases, neointimal hyperplasia occurred only in those vascular segments in which the IEL was ruptured. In all but two of the ballooninjured sites the underlying medial layer was also completely lacerated at the site of IEL rupture. The location of IEL rupture was primarily within the segment of vascular wall adjacent to the perivascular myocardium (Fig. 2). Because of the strong correlation between IEL rupture and resultant lesion formation, those ballooned segments with intact IELs (and thus no proliferation) were excluded from further analysis. Additionally, of the ballooned sites included for further analysis, only those representative cross-sections having an IEL rupture underwent morphometric evaluation (n = 2 to 6 sections per lesion site).

Representative LAD coronary arterial lesions induced by PTCA overinflation in domestic feeder pigs, high-lipid feeder pigs, and Yucatan minipigs are shown in Fig. 3. The lesions were consistently eccentric and localized to the region of IEL and underlying medial rupture. The neointimal lesions commonly extended through the normal medial layer to the external elastic lamina (EEL), which was consistently thinned in 15 of the 18 LAD sites examined from all three groups. The formed lesions also protruded to a limited extent into the luminal area and over adjacent segments of the vascular wall with intact IEL. Mild dissection flaps, such as those shown in Figs. 2, B, and 3, B were present in 61% of the balloon-injured sites. At these sites of medial dissection from the EEL, a narrow band of neointimal cells comprising < 6% (0.04 * 0.01 mm2/0.68 + 0.07 mm2, n = 9) of the total neointimal lesion area usually pervaded under the adjacent medial cell layer. Because dissection flaps were not present at all of the balloon-injured sites, any lesion located under the flap region was not included in the morphometric determination of neointimal area that was subsequently used to calculate the proliferation index of the lesion site (Fig. 1). Last, at all ballooned sites an endothelial cell-like layer covered the neointimal lesions that formed. The composition of the LAD neointimal lesions was similar in appearance in all experimental groups, consisting primarily of a loosely organized group of smooth-muscle cells interposed in extracellular matrix. Immunohistologic staining for a-smooth-muscle-cell actin suggested the abundance of smoothmuscle cells in the lesions (Fig. 4). Oil red 0 staining was performed on representative neointimal lesions

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et al. 25

Fig. 3. Histologic sectionsof porcine LAD coronary artery harvested 4 weeksafter balloon overinflation. A and 6, Uninvolved and overstretched regionsof coronary artery from feeder pig. Arrow, IEL; NI, neointima. C and D, Uninvolved and overstretched regionsof coronary artery from feederpig placedon high-lipid diet. E and F, Uninvolved and overstretched regionsof coronary artery from Yucatan minipig. (Original magnification X500.)

obtained from all three groups of pigs. No lipid deposition was observed in any of the lesions examined, including those obtained from feeder pigs placed on the high-lipid diet. In a subset of the above experiments, balloon overinflation was performed in the LCX coronary artery. The LAD and LCX vascular beds were sepa-

rated for study because of differences in the composition of periadventitial tissue surrounding the vessels (proximal LCX embedded primarily in adipose, LAD embedded approximately 50 % in epicardium). The success rates in achieving neointimal hyperplasia in either the LCX or its proximal branch ranged from 2 (40%) of 5 in both the normal diet and high-

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Fig. 4. Presenceof a-smooth musclecell actin, indicated by brown-red stain, in porcine LAD coronary artery harvested 4 weeksafter balloon overinflation. Paraffin sectionswereimmunostainedwith streptavidin biotin procedure in which an antibody-to-mouse Lu-smoothmusclecell actin wasused.Positive staining was present in both media (M) and neointimal focal lesion (NZJ. (Original magnification x500.) Fig. 5. Region of overinflated LCX coronary artery harvested from feeder pig 4 weeksafter coronary balloon overinflation. Neointimal focal lesion (NZ) originating at IEL fracture alsoextends along IEL (arrow). (Original magnification X500.) Fig. 6. Representative sectionof porcine LCX coronary artery showingacute damageinduced by coronary balloon overinflation. This segmentof ballooned artery was harvested 1 hour after balloon procedure in which ratio of inflated balloon diameter/artery diameter was1.18.This mechanicalinjury induced complete fracture of the IEL and media (arrows). (Original magnification x200.)

lipid diet feeder pig groups, to 3 (100 % ) of 3 (p = 0.09 vs other group frequencies, chi-square test) in the Yucatan minipig group. As observed in the LAD bed, neointimal lesions rich in smooth-muscle cells were present only where IEL rupture had occurred in the LCX ballooned sites (Fig. 5). Subsequent experiments were performed in a separate series of five feeder pigs to examine acutely the extent of vascular damage induced by the PTCA overinflation procedure. Six sites of coronary arterial balloon overinflation (three LAD regions, three LCX regions having no proximal branch overlap; overinflation ratio = 1.30 2 0.08) were harvested within 1 hour of PTCA. Complete or nearly complete endothelial denudation with resultant IEL rupture and underlying medial damage was observed in every case (Fig. 6). In these experiments, acute medial damage ranged from severe compression (1 of 6 cases) to complete severing of the cell layer (5 of 6 cases).

Morphometry. Table II summarizes the extent of arterial overstretch, the severity of IEL rupture (injury index), and the extent of neointimal hyperplasia (proliferation index) induced by balloon overinflations in the LAD vascular beds of the three groups of pigs under study. Mean overinflation ratios calculated at the balloon injured sites were 1.30 f 0.06 in the feeder pig group, 1.41 f 0.07 in the high-lipid feeder pig group, and 1.19 k 0.05 in the Yucatan minipig group; they were not statistically different from each other. Additionally, there were no significant differences between groups in the actual diameters of the preballooned arterial segments as determined from control angiograms. The degree of overinflation applied to the porcine LAD coronary arteries caused similar magnitudes of IEL rupture between groups, with the lengths of IEL rupture ranging from 17.4% Z!Z2.1% to 18.7 % i 2.1% of the total IEL circumference. Supplementing the feeder pig diet

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with cholesterol and lard before and during the PTCA procedure did not influence the magnitude of neointimal hyperplasia associated with this vascular damage because the proliferation index of this group (NI/M = 0.80 f 0.15) was indistinguishable from that observed in feeder pigs kept solely on a normal diet (0.73 f 0.09). In contrast, there was a trend toward greater LAD neointimal hyperplasia in the Yucatan minipig group because the proliferation index of this group was 56 % greater than that measured in the feeder pig-normal diet group (1.14 vs 0.73, p = 0.07). Morphometric analysis was also performed on the limited number of the balloon-injured LCX sites that showed neointimal hyperplasia. Direct comparison of the LCX injury and proliferation indexes measured in the Yucatan minipig (n = 3) and combined feeder pig groups (f high-lipid diet, n = 4) revealed no marked differences in terms of the extent of IEL rupture (injury index = 22.2% f 6.4% vs 28.3% f 4.2%, p = not significant [NS]) or the degree of neointimal hyperplasia that developed (proliferation index = 1.47 f 0.66 vs 1.66 i 0.41,~ = NS). In three of the seven LCX lesion sites, the overinflation ratios applied at the time of balloon injury were not determined because of the presence of a large diagonal branch whose image on visualization with one-plane fluoroscopy overlapped that of the main LCX. Although preventing determination of group mean overinflation ratios, we ensured that the applied overinflation ratio was > 1.0 in these casesby choosing an arterial site where the overlapping image was smaller than that of the inflated balloon catheter. Comparison of the overall morphometric data obtained from the LCX balloon-injured sites (n = 7) with that collected from the LAD (n = 18 sites) revealed a significant trend toward greater injury in the LCX vascular beds (injury index = 25.7% ? 3.5% [LCX] vs 18.2% +- 1.2% [LAD], p < 0.05) that correlated directly with a greater degree of hyperplasia (proliferation index = 1.58 * 0.22 [LCX] vs 0.90 _+0.10 [LAD], p < 0.05). Regardless of the experimental group or coronary vessel that was overinflated, there was a positive correlation between the extent of IEL rupture and the resultant degree of neointimal proliferation. Because the slopes of the regression lines calculated for each group and each vascular bed were not statistically different, a regression analysis between the injury index and the proliferation index was performed by using all of the PTCA overinflation sites. Fig. 7 illustrates the strong relationship that existed between the calculated injury and proliferation indexes (r = 0.74, p < 0.05)

et al.

27

Table II. Morphologic examination of porcine LAD coronary arteries exposedto intentional PTCA overinflation Injury Group Domestic feeder pigs Normal diet (n = 7) High-lipid diet (n = 4) Yucatan minipigs (n = 7)

Ouerinflation ratio*

Proliferative response (NIIM)

index (ZELfIlEL, x 100)

1.30 + 0.06 1.41 + 0.07

0.73 + 0.09 0.80 * 0.15

17.4 + 2.1 18.5 k 2.8

1.19 + 0.05

1.14 t 0.21

18.7 + 2.1

Values are mean t SEM. LAD, Left anterior descending; PTCA, percutaneous transluminal coronary angioplasty; NI/M, neointimal lesion area/ medial area; IEL#EL,, internal elastic lamina fracture length/IEL circumference. *Degree of overinflation quantitated as ratio of inflated balloon diameter to control vessel diameter as determined from angiographic recordings.

in this model of coronary artery neointimal hyperplasia. Serum lipid profile. Table III summarizes the serum lipid profiles that were periodically determined in the pigs during the experimental protocol. At the time of PTCA, serum levels of HDL, triglycerides, and total lipids were quite similar in both feeder pigs (normal diet) and Yucatan minipigs, whereas total cholesterol levels in the feeder pig tended to be higher (75 + 3 mg/dl vs 58 f 5 mg/dl for the Yucatan group, p = 0.08). Serum lipid profiles were monitored more extensively in those pigs fed a diet containing cholesterol and lard. After 30 to 40 days of this high-lipid diet, serum levels of total cholesterol, HDL, total triglycerides, and total lipids in these pigs had risen by 2.3-, 1.6-, 1.5-, and 2.4-fold, respectively. Indeed, at the time of PTCA, the serum level of every parameter measured was significantly higher in the high-lipid diet group versus those monitored in either the normal-diet feeder pig or Yucatan mini-pig groups. Within 3 weeks of terminating the lipid-supplemented diet, however, the serum lipid profile of the high-lipid diet group wasreturning toward prediet levels. Hemodynamics and body weight gains. Blood pressure and heart rate data monitored before the PTCA overinflation procedure and accompanying body weight data are summarized in Table IV. Between groups there were no significant differences in prePTCA mean arterial pressure or heart rate in the anesthetized pigs. However, significant group differences did exist in terms of the percentage of body weight increases observed over the 4-week recovery

28

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r=0.74, 2.4

-

2.2

-

January 1994 Heart Journal

‘~(0.05

0

2 1.8

-

1.6

-

1.4

+

1.2 1 0.8

-

0.6

-

0.4

-

0.2

-

0

0 0

.

I

0 5

I

I

Fig. 7. Positive correlation between severity of neointimal (NI) proliferation (proliferation index; LAD and LCX coronary arteries (n = 18) 4 weeks descending artery; LCX, left circumflex coronary

Table

Ill. Serum

lipid

levels in pigs undergoing

At PTCA Prediet At PTCA At sacrifice At PTCA

4)

Yucatan minipigs (n = 4) Values are mean + SEM in milligrams *p < 0.05 vs corresponding time value tp < 0.05 vs prediet value in high-lipid $p < 0.05 YS sacrifice value in high-lipid

Table

IV. Control

and body weight Mean arterial pressure (mm f&d

Domestic feeder pigs Normal diet (n = 7) diet

Yucatan minipigs Values are mean k SEM. *p < 0.05 vs corresponding tp < 0.05 vs corresponding

I 35

in domestic in high-lipid

feeder pig-normal diet group.

I 40

overinflation

Total cholesterol

75 f

3

33 f

2

31 f 3 50 2 2*t$ 38 + 2

58 i- 5 lipoprotein; diet and

Total triglyceride

HDL

76 f 12 175 + 16*t$ 80 -+ 5

33 + 5

PICA, Yucatan

Total

33 * 4

183

71 + 10 104 + 43*$ 45 2 7

163 399 202

39

+ 5

percutaneous transluminal minipig groups.

148 coronary

lipids

k 12 + 19 f 48*t$ rt 11 f 22

angiography

gains Body Heart rate (beatslmin)

70 + 3 71 t 3 90 i: 10

(n = 4) (n = 7)

value value

I

vascular injury (injury index; see Fig. 1) and extent of see Fig. 1) that developed in feeder and Yucatan minipig after balloon overinflation. M, Media; LAD, left anterior artery.

per deciliter. HDL, High-density within domestic feeder pig-normal diet group. diet group.

hemodynamics

GFOU~S

High-lipid

PTCA

Time period

Group

Domestic feeder pigs Normal diet (n = 5) High-lipid diet (n =

I

10

99 111 98

diet

group.

k 6 * 7 + 8

Day

22.4 21.0 26.1

1

c 1.5 * 1.5 IL l.l*t

weight

(kg) Day

40.6 40.9 34.9

28

+ 2.3 + 2.3 * 1.5*

% Increase in body weight

81.6 95.8 33.5

2 4.1 ? 4.5* IL 2.7*t

Volume 127, Number 1 American Heart Journal

period. Compared with the feeder pigs and high-lipid diet feeder pigs that gained on average between 18.2 and 19.9 kg/pig (82 % to 96 % increase in body weight) during the recovery period, the average weight gain in the Yucatan minipigs was significantly less (8.8 kg/pig or 33.5% increase in body weight; p < 0.05 vs other groups). DISCUSSION

Although intentional balloon overinflation of coronary arteries has previously been shown to induce intimal hyperplasia in pigs, the relative unpredictability and deficiency of this response in comparison to that obtained by using endoluminal stents has diminished interest in balloon overinflation as a viable model of coronary artery restenosis.16>l8 In this study we have extended these earlier observations by demonstrating that neointimal hyperplasia proliferation is consistently achieved (>85% success in the LAD vascular bed of surviving pigs) in overstretched coronary arteries within 4 weeks of PTCA. Comparison of ballooned regions of porcine coronary artery harvested either acutely (Fig. 6) or chronically (Fig. 2) after the PTCA procedure strongly indicates that the formed neointimal lesions were reparative in nature because they primarily filled the void left in the vessel wall at the point of IEL and medial disruption. Consistent with this observation was the strong positive correlation that existed between the percentage of IEL circumference that ruptured and the magnitude of the resultant intimal hyperplastic response. This direct correlation between severity of IEL-medial damage and extent of intimal hyperplasia was also observed by Schwartz et al.16T26in their oversized stent model of porcine coronary artery restenosis. On the basis of this relationship it is apparent that incorporation of an injury index analysis when evaluating the morphometric data from these restenosis animal models is extremely important to avoid the generation of false-positives or -negatives when testing new antirestenotic therapies. The induction of intimal hyperplasia responsesvia balloon overinflation was dependent on rupture of the IEL and with it, extensive medial damage and EEL thinning. This relationship between the dependence of IEL damage and intimal hyperplasia has been observed in other balloon-injury experiments in porcine coronary arteries where PTCA catheters were used.16>I8 Similarly, restenotic lesions associated with IEL rupture and underlying medial damage have also been observed in human PTCA patients, although IEL rupture is not always a requisite for the clinical occurrence of restenosis.42lg In other pig models of restenosis, intimal hyperplastic re-

Humphrey

et al.

29

sponses are observed in either ballooned carotid arteriesi5 or deendothelialized coronary arteries2’ despite preserved IEL integrity. Compared with these other studies, the dependency of IEL rupture for the induction of neointimal hyperplasia by balloon overinflation may be related to the shortened region of intimal damage (20 mm vs 30 to 40 mm) and/or reduced mural thrombus formation at the damaged site. We used low oral dosesof aspirin throughout the recovery period to mimic common clinical practice.2 This treatment proved effective throughout the protocol because there was little angiographic evidence of extensive thrombosis at the ballooned site either acutely or chronically after PTCA. Additionally, morphologic examination did not reveal the presence of mural thrombi in the neointimal regions. Oral aspirin doses as low as 1 mglkglday have been shown to be effective in reducing platelet deposition in regions of deep arterial injury in pigs.27 The inherent thrombogenic responses of porcine arteries to balloon angioplasty15 and the propensity for fatal arrhythmias during cardiac procedures in pigs23were of major concern to us as we embarked on this study. Therefore, in addition to aspirin, a number of agents including heparin, verapamil, bretylium, and nitroglycerin, were used as standard protocol to improve the survival of pigs undergoing coronary artery overinflation. The 25 % mortality rate reported for this model may be artifically high, asthe incidence of death resulting from coronary artery dissection and ventricular fibrillation became less frequent on awareness of the limits to which the vessels could be successfully overinflated and of the need to reduce the total intracoronary dose of nitroglycerin in those animals in which coronary artery perfusion pressure was compromised by placement of the coronary guide catheter. A major question that we addressed in this study was whether the neointimal hyperplastic responses induced by balloon overinflation would be affected by the sexual maturity of the pig. The 2- to 3-month-old domestic feeder pig is commonly used in experimental models of restenosis because of its ready access, low initial cost, and manageable size at this stage of development. 15,16,l8 In contrast to these juvenile pigs, the 5- to B-month-old Yucatan minipig has reached sexual maturity and has already approached its maximal skeletomuscular size.28,2gThus it could be theorized that the adult Yucatan minipig would present a more clinically relevant subject to evaluate PTCA-induced restenosis. In the present study there was a trend (p = 0.07) toward larger neointimal proliferative lesions in the ballooned LAD coronary arteries of the Yucatan minipig versus those observed

30

Humphrey

et al.

in the domestic feeder pigs, despite equivalent vascular injury. However, in the more severely damaged LCX coronary arteries where the mean vascular injury index was roughly 40% greater than that measured in the LAD, the proliferation index was similar in both experimental groups. These observations suggest that in this model any differences in the vascular hyperplastic responses induced in sexually mature Yucatan minipigs and juvenile feeder pigs are modest and more likely to appear after moderate vascular injury. A high-lipid diet feeder pig group was introduced in this study to begin the evaluation of the effect of diet manipulation on the vascular injury response induced by balloon overinflation. In these experiments we chose to terminate the high-lipid diet at 7 days after ballooning, a time interval previously shown to be required for complete regrowth of endothelial-like cells after balloon overinflation of procine carotid arteries.5TI5 Despite elevating total serum cholesterol from a control level of 76 k 12 mg/dl to a level of 175 f 16 mg/dl at the time of PTCA, a concurrent high-lipid diet did not amplify the intimal hyperplastic response to balloon injury. Several factors may have contributed to this finding. First, the effect of elevated cholesterol and lipid level on smooth-muscle proliferation and migration during the early stages of the restenotic process may have been negated by the 1.6-fold rise in serum levels of HDL. Although not firmly established, there is growing clinical evidence that elevated HDL levels are associated with reduced rates of coronary artery restenosis after various catheter interventions used to treat coronary vascular disease.30Second, the cholesterol level achieved with the high-lipid diet may have been too low, and the duration of hyperlipidemia too short, to have a marked effect on lesion size. In porcine models of atherosclerosis, lipid-laden foam cells can be found in neointimal arterial lesions if total serum cholesterol levels are severely elevated (1400 mg/dl) for an extended period of time (13 months).22, 31It is conceivable that by extending the period of the high-lipid diet for the duration of the 4-week recovery period, we may have had a more dramatic effect on the neointimal proliferative responses that ultimately developed. However, comparison of results obtained in stent models of coronary artery restenosis in hyperlipidemic32 and normal pig@ suggest, as our results do, that the magnitude of the hyperplastic response induced within 4 weeks of balloon injury may be more dependent on the extent of medial injury rather than the lipid profiles that are achieved by diet manipulation.

American

January 1994 Hearl Journal

The intentional overinflation of healthy arteries to induce intimal hyperplasia responses may more correctly be defined as a primary model of vascular injury rather than a model of restenosis per se. Nonetheless, the myofibrotic lesions that developed were similar in appearance and composition to the coronary arterial lesions that develop after PTCA in human beings. Endoluminal stent models of porcine coronary artery restenosis do produce much larger lesions than those induced by balloon overinflation. However, in these stent models chronic placement of the metallic scaffolding elicits a marked foreign body inflammatory response and a persistent, circumferential tension against the luminal surface of the artery.16, i8 These stimuli may overwhelm the potential efficacy of antirestenotic agents. This concern is alleviated when the proliferative responses are elicited by simple balloon overinflation. Small animal models of restenosis such as the rat carotid balloon injury model and the hypercholesterolemic rabbit iliac balloon injury model have proven valuable in defining the events leading up to restenosis and in the initial screening of potential antirestenotic agents.g-ll, 13,l4 H owever, because of limitations in ‘predicting restenosis in human beings, the effectiveness of agents in minimizing restenosis in these small animal models has not translated well to the clinical setting. l1 In this light the porcine model of restensosis described here is not without limitations, that is, the requirement of IEL rupture to induce a neointimal response, the relatively short recovery period, and the ballooning of healthy rather than diseased coronary arteries. Nonetheless, implementation of such a secondary large animal model of restenosis to confirm the antirestenotic activity of new agents and/or regimens may prove beneficial in improving the predictability of their clinical efficacy. The value of follow-up testing in large animal models was recently illustrated in the preclinical development of the angiotensin-converting enzyme inhibitor cilazapril, which was shown to be an effective antiproliferative agent in the rat33 but ineffective in reducing restenosis in humans.34 Subsequent evaluation of cilazapril in a porcine model of restenosis failed to support the positive findings in the rat.35 In conclusion, this study demonstrates that coronary artery balloon overinflation can effectively induce consistent and quantifiable neointimal lesions in either Yucatan minipigs or feeder pigs. Implementation of this porcine model before any planned clinical trial may be useful in confirming the antirestenotic effects of agents previously evaluated in smaller animal models.

Volume 127, Number 1 American Heart Journal

We thank manuscript.

Jane

Humphrey

L. Johnson

for preparation

of the text

in the 19.

REFERENCES 1. Muller

2.

3.

4.

5.

6.

I.

8.

9. 10.

11.

12. 13.

14.

15.

16.

17.

18.

DWM, Ellis SG, Top01 EJ. Experimental models of coronary artery restenosis. J Am Co11 Cardiol 1992;19:41832. Serruys PW, Luijten HE, Beatt KJ, Geuskens R, de Feyter PJ, van den Brand M, Reiber JHC, ten Katen HJ, van Es GA, Hugenholtz PG. Incidence of restenosis after successful coronary angioplasty: a time-related phenomenon. A quantitative angiographic study in 342 consecutive patients at 1,2,3, and 4 months. Circulation 1988;77:361-71. Leimgruber PP, Roubin GS, Hollman J, Cotsonis GA, Meier B, Douglas JS, King SB III, Gruentzig AR. Restenosis after successful coronary angioplasty in patients with single-vessel disease. Circulation 1986;73:710-7. Waller BF. Crackers, breakers, stretchers, drillers, scrapers, shavers, burners, welders, and melters-the future treatment of atherosclerotic coronary artery disease? A clinical-morphologic assessment. J Am Co11 Cardiol 1989;13:969-87. Chesebro JH, Lam JYT, Badimon L, Fuster V. Restenosis after arterial angioplasty: a hemorrheologic response to injury. Am J Cardiol 1987;60:10-6B. Harker LA. Role of platelets and thrombosis in mechanisms of acute occlusion and restenosis after angioplasty. Am J Cardiol 1987;60:20-8B. In JH. Fuster V. Israel D. Badimon L. Badimon J. Chesebro jH. The role of platelets,‘thrombin, and hyperplasia in restenosis after coronary angioplasty. J Am Co11 Cardiol 1991; 17:77-88B. Liu MW, Roubin GS, King SB III. Restenosis after coronary angioplasty: potential biological determinants and role of intimal-hyperplasia. Circulation 1989;79:1374-87. Clowes AW. Reidv MA. Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest 1983;49:208-15. Clowes AW, Schwartz SM. Significance of quiescent smooth muscle cell migration in the injured rat carotid artery. Circ Res 1985;56:139-45. Hermans WRM, Rensing BJ, Strauss BH, Serruys PW. Prevention of restenosis after percutaneous transluminal coronary angioplasty: the search for a magic bullet. AM HEART J 1991;122:171-87. Popma JJ, Califf RM, Top01 EJ. Clinical trials of restenosis after coronary angioplasty. Circulation 1991;84:1426-36. Faxon DP, Sanborn TA, Weber VJ, Haudenschild C, Gottsman SB, McGovern WA, Ryan TJ. Restenosis following transluminal angioplasty in experimental atherosclerosis. Arteriosclerosis 1984;4:189-95. Sarembock IJ, LaVeau PJ, Sigal SL, Timms I, Sussman J, Haudenschild C, Ezekowitz MD. Influence of inflation pressure and balloon size on the development of intimal hyperplasia after balloon angioplasty: a study in the atherosclerotic rabbit. Circulation 1989;80:1029-40. Steele PM, Chesebro JH, Stanson AW, Holmes DR Jr, Dewanjee MK, Badimon L, Fuster V. Balloon angioplasty: natural history of the pathophysiological response to injury in a pig model. Circ Res 1985;57:105-12. Schwartz RS, Murphy JG, Edwards WD, Camrud AR, Vlietstra RE, Holmes DR. Restenosis after balloon angioplasty: a practical proliferative model in porcine coronary arteries. Circulation 1990,82:2190-200. French JE, Jennings MA, Florey HW. Morphological studies on atherosclerosis in swine. Ann N Y Acad Sci 1965;127:78099. Karas SP, Gravanis MB, Santoian EC, Robinson KA, Anderberg KA, King SB III. Coronary intimal proliferation after

20.

21.

22.

23.

24.

25.

26.

27.

28. 29.

30. 31.

32.

33.

34.

35.

et al.

31

balloon injury and stenting in swine: an animal model of restenosis. J Am Co11 Cardiol 1992;20:467-74. Gravanis MB, Roubin GS. Histopathologic phenomena at the site of percutaneous transluminal coronary angioplasty: the problem of restenosis. Hum Path01 1989;20:477-85. Arakawa H, Mutoh S, Hirosumi J, Yamaguchi I. Effect of nilvadipine on balloon catheterization-induced intimal thickening of the coronary artery in miniature pigs. J Cardiovasc Pharmacol 1992;20:731-4. Roubin GS, Cannon AD, Agrawal SK, Macander PJ, Dean LS, Baxley WA, Breland J. Intracoronary stenting for acute and threatened closure complicating percutaneous transluminal coronary angioplasty. Circulation 1992;85:916-27. Gerrity RG, Naito HK, Richardson M, Schwartz CJ. Dietary induced atherogenesis in swine: morphology of the intima in prelesion stages. Am J Path01 1979;95:775-92. Rodgers GP, Cromeens DM, Minor ST, Swindle MM. Bretylium and diltiazem in porcine cardiac procedures. J Invest Surgery 1988;1:321-6. Knight JA, Anderson S, Rawle JM. Chemical basis of the sulfo-phospho-vanillin reaction for estimating total serum lipids. Clin Chem 1972;18:199-202. Skalli 0, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against cr-smooth muscle cell actin: a new probe for smooth muscle cell differentiation. J Cell Biol 1986;103:2787-96. Schwartz RS, Huber KC, Murphy JG, Edwards WD, Camrud AR, Vliestra RE, Holmes DR. Restenosis and the proportional neointimal response to coronary artery injury: results in a porcine model. J Am Co11 Cardiol 1992;19:267-74. Lam JYT, Chesebro JH, Steele PM, Heras M, Webster MWI, Badimon L, Fuster V. Antithrombotic therapy for deep arterial injury by angioplasty: efficacy of common platelet inhibition compared with thrombin inhibition in pigs. Circulation 1991;84:814-20. Swindle MM, Smith AC, Hepburn BJS. Swine as models in experimental surgery. J Invest Surgery 1988;1:65-79. Gal D, Rongione AJ, Slovenkai GA, DeJesus ST, Lucas A, Fields CD, Isner JM. Atherosclerotic Yucatan microswine: an animal model with high-grade, fibrocalcific, nonfatty lesions suitable for testing catheter-based interventions. AM HEART J 1990;119:291-300. Austin GE. Lipids and vascular restenosis. Circulation 1992;85:1613-5. Griggs TR, Bauman RW, Reddick RL, Read MS, Koch GG, Lamb MA. Development of coronary atherosclerosis in swine with severe hypercholesterolemia: lack of influence of von Willebrand factor or acute intimal injury. Arteriosclerosis 1986;6:155-65. Buchwald AR, Unterberg C, Nebendahl K, Grone H-J, Wiegand V. Low-molecular-weight heparin reduces neointima1 proliferation after coronary stent implantation in hypercholesterolemic minipies. Circulation 1992:86:531-7. Powell JS, Clozel JP,-Muller RKM, Kuhn H, Hefti F, Hosang M, Baumgartner HR. Inhibitors of angiotensin-converting enzyme prevent myointimal proliferation after vascular injury. Science 1989;245:186-8. The Multicenter European Research Trial With Cilazapril After Angioplasty to Prevent Transluminal Coronary Obstruction and Restenosis (MERCATOR) Study Group. Does the new angiotensin-converting enzyme inhibitor cilazapril prevent restenosis after percutaneous transluminal coronary angioplasty? Results of the MERCATOR Study: a multicenter, randomized, double-blind, placebo-controlled trial. Circulation 1992;86:100-10. Lam JYT, Lacoste L, Bourassa MG. Cilazapril and early atherosclerotic changes after balloon injury of porcine carotid arteries. Circulation 1992;85:1542-7.