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Proliferative and Lipid Metabolism Response to Balloon Angioplasty inCanine Renal Arteries

CESARE ORLANDI, MD, JAI PAL SINGH, PHD, FRANK P. BELL, PHD,

ROBERT G. SCHAUB, PHD

Kalamazoo, Michigan

The mechanisms responsible for reocclusion after percuta­neous transluminal angioplasty are still poorly understood.The effects of angioplasty on arterial morphology, deoxyri­bonucleic acid (DNA) synthesis eH-thymidine incorpora­tion) and lipid metabolism e4C-oleate incorporation) werestudied in renal arteries of 24 male mongrel dogs. Balloon­dilated (identified by Evans blue dye accumulation) andadjacent normal arterial segments were collected 90 minand 2, 5 and 14 days after the procedure. The immediatevascular response was endothelial cell denudation andplatelet accumulation.

Two weeks after angioplasty, healing of the luminalsurface by "endothelial-like" cells, mild smooth muscle cellproliferation and an angiogenic response with capillarygrowth into the media were observed. DNA synthesis was

Percutaneous transluminal angioplasty has become an im­portant adjuvant and in many cases a viable alternative tovascular surgery in treating narrowed or occluded arteries(1). There are advantages to angioplasty compared withsurgery; however, restenosis is a major limitation of thistechnique. Different institutions have reported reocclusionincidence rates within 6 months from the procedure varyingbetween 16% and 45% (2-7). The mechanisms that deter­mine restenosis and occlusion after balloon angioplasty arestill poorly defined. A role for platelet adhesion and throm­bus formation in both acute occlusion (8) and restenosis (9)has been demonstrated. However, very little is known aboutthe changes that angioplasty produces on the vessel wall.The experiments described in this study were designed tocharacterize the time-related changes induced by renal ar­tery balloon dilation on vessel wall morphology, arterial lipidmetabolism and deoxyribonucleic acid (DNA) synthesis.

From The Upjohn Company, Kalamazoo, Michigan.Manuscript received July 10, 1989; revised manuscript received Decem­

ber 13, 1989, accepted January 5, 1990.Address for reprints: Robert G. Schaub, PhD. Genetics Institute, 87

Cambridge Park Drive, Cambridge, MA 02140-2387.

© 1990 by the American College of Cardiology

increased in balloon-dilated segments at day 5 comparedwith adjacent nonballoon-dilated artery. This increase inDNA synthesis persisted in the 2 week postangioplastysegments. Additionally, angioplasty produced both quanti­tative and qualitative changes in arterial lipid synthesis.The most dramatic change was an increase in sterol ester­ification that was apparent as early as 90 min after angio­plasty; the change persisted through day 5 but diminishedtoward baseline by day 14.

Angioplasty-induced alterations of arterial metabolismparallel aspects of the atherogenic process and may beinvolved in the pathogenesis of postangioplasty reocclusion,particularly in the presence of additional risk factors, suchas hyperlipemia.

(J Am Coil CardioI1990;15:1394-400)

MethodsRenal angioplasty procedure. Twenty-two male mongrel

dogs weighing between 18 and 30 kg were anesthetized withsodium pentobarbital (30 mg/kg body weight intravenously).After tracheal intubation, respiration was maintained with aHarvard model 607 animal respirator. The animals wereheparinized with a bolus of 100 USP units/kg intravenously.Angioplasty of both renal arteries was performed with aCordis polyethylene balloon catheter (11 mm x 1 em)inserted through a right femoral arteriotomy. This catheterwas selected because of the relatively short segment of mainrenal artery available before its distal branching. The cath­eter was advanced under fluoroscopic guidance and selec­tively positioned immediately distal to the bifurcation of therenal arteries. Both renal arteries were dilated (maximaldilation 11 mm) by five 30 s inflations, with 60 s intervalsbetween inflations. The average diameter of the renal arter­ies was 5 to 7 mm. No acute complications such as dissectionor thrombosis occurred as a result of dilation.

Renal artery processing. Renal arteries were collected 90min (n = 3), 48 h (n = 6), 5 (n = 6) and 14 (n = 7) days afterangioplasty. Evans blue dye solution in Tyrode buffer

0735-1097/90/$3.50

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Figure 1. Ninety minutes after bal­loon dilation, scanning microscopyrevealed a layer of adherent plate­lets (P) on the Evans blue-stainedregion of the renal arteries (A andB). Leukocytes (L) were also foundassociated with the platelets. Ini­tially, this association appearedrandom (B). However, after 2days,leukocytes were located at the in­terface of the dilated regions withthe nondilated region as demar­cated by Evans blue dye uptake(C). D, Typical renal artery seg­ment adjacent to the dilated regionsthat did not accumulate Evans bluedye. Bars represent 10 ~m.

(pH 7.4) was injected intravenously (2 ml/kg) and allowed tocirculate for 1 h before tissue collection. The uptake ofEvans blue dye by the balloon-dilated areas of the renalartery allowed demarcation of the injured from adjacentnonballoon-dilated areas. The left renal artery was surgicallyremoved for DNA synthesis and arterial metabolism evalu­ation as described later. The dogs were then injected with alethal dose of intravenous potassium chloride (5 mI). Theright renal artery was perfused anterograde with Tyrodesolution until the perfusate was cleared of blood and thenfixed for I h at a pressure of 120 mm Hg with 1% glutaral­dehyde in Tyrode solution. The artery was removed andfurther processed for scanning and transmission electronmicroscopy as described below. All studies conformed to theposition of the American Heart Association on researchanimal use.

Histopathology. The dilated portion of the renal arterywas easily identified by the uptake of Evans blue dye, whichproduced a dark to light blue band at the site of injury.Nonballoon-dilated areas (unstained) and balloon-dilatedareas (blue-stained segments) were further processed forscanning and transmission electron microscopy. Samples forscanning electron microscopy were prepared as previouslydescribed (10). Fixed tissues were dehydrated by criticalpoint drying with a Denton vacuum DCP-l critical pointdryer. The samples were mounted on aluminum stubs andcoated with gold (approximately 200 to 220 a) in a HummerX sputter coater. Each sample was examined with a JEOL300 scanning electron microscope for appearance of the

endothelial cells and adhesion of blood cells to the lumen ofthe artery.

One millimeter segments from unstained and blue-stainedareas were cut in longitudinal sections and prepared fortransmission electron microscopic examination as previ­ously described (11). Fixed tissue was embedded in Araldite/Medcast resin (Ted Pella). Both thick and thin sections weremade on an LKB NOVA ultrotome. Thick sections werestained with Toluidine blue. Thin sections were stained withan LKB ultrastainer with uranyl acetate (1 h) and lead citrate(15 min). Thin sections were examined with a Philips 201electron microscope.

Arterial lipid metabolism. After their surgical removal,the left renal arteries were immediately rinsed in chilled0.9% sodium chloride solution and then bisected longitudi­nally. One of the longitudinal segments was further subdi­vided into its "injured" and "noninjured" portions (basedon Evans blue staining patterns) for analysis of lipid metab­olism. The tissues were incubated at 37°C for 90 min in 3 mlof mixture of Medium 199 (Gibco) and normal dog serum(1: I, v/v) (pH 7.4) that contained 1 p.Ci 14C-oleate/ml (Du­Pont-NEN, specific activity 57.0 Ci/mol). For purposes ofmaintaining consistency in the incubations, an amount ofincubation medium sufficient to complete the study wasprepared. The medium was stored frozen in aliquots thatwere thawed immediately before their use and before theaddition of the 14C-oleate substrate. After incubation, thetissues were freed of adventitial debris, and their lipids wereextracted by homogenization in chloroform/methanol mix-

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Figure 2. Endothelial cell migration to the denuded areas wasobserved in the 5 day, postdilation samples (A). However, largeportions of the luminal surfaces still had platelet/leukocyte adhesion(B). Bar represents 20 JLm in A and 5 JLm in B.

tures (12). A portion of each lipid extract was fractionated bythin layer chromatography to recover the individual lipidclasses, and the fractionated lipids were analyzed for radio­activity by liquid scintillation counting (12).

To assess the possibility that differences in lipid metabo­lism might exist a priori at different anatomic sites, we alsocompared lipid metabolism in segments of normal renalarteries of three dogs that did not undergo angioplasty.Segments were selected to approximate the location ofballoon dilated- and nonballoon-dilated segments from thedogs that underwent angioplasty.

Arterial DNA synthesis. The remaining segment from thebisected left renal artery was used for assessment of DNAsynthesis in the arterial wall. Tissue sections (15 to 20 mgwet weight) were incubated in 2 ml Dulbecco's modifiedEagle's medium, containing 10 mCi/ml 3H-thymidine for 4 hat 37°C under 95% air and 5% carbon dioxide. At the end ofthe incubation, the tissues were washed in phosphate-

buffered saline solution followed by 5% trichloroacetic acidand then once again in phosphate-buffered saline solution.The tissues were then solubilized in 0.5 ml Protosol, andradioactivity was determined by scintillation counting. Forautoradiography, sections were fixed immediately after the4 h incubation in 5% formalin. Eight to 10 serial thicksections were cut and mounted on glass cover slides.

Statistical analysis. Values are expressed as mean values± SEM as index of dispersion. Statistical analysis wasperformed using Student's t test for paired variates. The nullhypothesis was rejected with p < 0.05.

ResultsHistopathology (Fig. 1 to 4). Scanning electron micro­

scopic analysis of the balloon dilated areas showed com­pletely denuded endothelium replaced by a layer of adherentplatelets within 90 min after dilation (Fig. 1). Adjacentnonballoon-dilated areas had intact endothelium and no celladhesion. Leukocytes were also found associated with plate­lets on the balloon-dilated areas. Ninety minutes after an­gioplasty, the leukocyte adhesion was random. However, 2days after angioplasty, leukocytes were found at the borderareas between intact endothelium and denuded surface.These leukocytes were adherent to both platelets and endo­thelial cells. Five days after angioplasty, endothelial cellmigration to the denuded areas was observed (Fig. 2). Thoseareas that were still denuded had a layer of adherent plate­lets, some of which appeared to have recently attached tothe lumen. Two weeks after angioplasty, much of the dilatedarea was covered with a layer of intimal cells that appearedto be of endothelial origin (Fig. 3). The orientation of thosecells was much more random than that of the typi<,:alluminalendothelial cells. In addition, shape, size and intercellularcontact were different from those of endothelium typical tothose arteries. Platelets and leukocytes were not adherent tothese neointimal cells. The only areas with platelet/leukocyte attachment were those few areas that still hadexposed basement membrane.

The most interesting finding was the observation thatmany areas ofthe media contained budding ofnew capillaryvessels (Fig. 4). The origin of these vessels was, most likely,the adventitia. In addition, as would be expected, bothmedial and intimal proliferation of smooth muscle cells wasobserved. The extent of proliferation into the intima wasinconsistent, but the media of dilated areas contained severallayers of smooth muscle cells that were decidedly abnormalin both morphology and orientation.

Arterial lipid metabolism (Fig. 5 and 6). Balloon angio­plasty modified the normal lipid metabolic profile of the renalartery. Ninety minutes and 2, 5 and 14 days after theprocedure, the injured tissue segments demonstrated anenhanced synthesis of total lipids relative to their contiguousnoninjured segments. The increased lipid synthesis was

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Figure 3, After 2 weeks, large por­tions of the balloon-dilated areaswere covered with a layer of intimalcells that were "endothelial-like" (Aand B). However, the cell to cellcontact, orientation and surface ap­pearance of these cells were signifi­cantly different from those of nonin­jured endothelium (see Fig. 1D). Insome areas, raised intimal lesionswere observed (C). Platelet accumu­lation was still evident in areas thatdid not have an intimal cell cover (D).Bars represent 5 JLm in Aand D, 10JLm in Band 100 JLm in C.

apparent at 90 min (approximately 60% increase) and con­tinued to increase until 5 days after angioplasty (threefoldincrease). At 14 days, the increase had diminished slightlybut remained at a level 80% above that of the noninjuredtissue (Fig. 5). We observed a trend (not statistically signif­icant) toward increased lipid synthesis at 2 and 5 days afterballoon dilation in the noninjured segments of the renalarteries. However, one can easily envision that ballooninflation would generate, to some extent, stress or stretch onthe contiguous arterial tissue that we designated as unin­jured. Clearly the "stretch" phenomenon is mild relative toangioplasty, because lipid synthesis is normal at 14 days inthe region adjacent to the angioplasty site.

Renal artery tissue collected from the three control dogsshowed no evidence of inherent differences in lipid metabo­lism based on anatomic location comparable with the dilatedand nondilated tissue from the dogs that underwent angio­plasty. Within each artery, the total incorporation of'4C-oleate into complex lipids (Fig. 5) and the percentdistribution of '4C-oleate into the phospholipid, diglycerideplus triglyceride and cholesteryl ester fractions were thesame (Fig. 6). As would be anticipated from the relativelylow levels of acyl CoA:cholesterol acyltransferase (ACAT)activity in normal artery (13,14), the proportion of 14C-oleateincorporated into cholesteryl esters was considerably lessthan «5%) that appearing in the other lipid fractions.

On fractionation of the lipids (Fig. 6), it appears that theinjury-induced stimulation of lipid synthesis consists of twodistinct components. The first of these is represented by a

generalized increase in synthesis of each of the three frac­tions (i.e., phospholipids, glycerides and cholesteryl esters).However, increases of phospholipids and glycerides were inthe range of 1.5- to twofold, whereas there was a fivefoldincrease in cholesteryl ester synthesis.

Arterial DNA synthesis (Fig. 7). Deoxyribonucleic acid(DNA) synthesis in the balloon-dilated artery was similar tothat of adjacent nondilated areas in the arteries studied 90min after angioplasty. Balloon-dilated segments collected onday 2 showed significantly higher DNA synthesis than didadjacent nonballoon-dilated areas. On days 5 and 14 afterangioplasty, the DNA synthesis response in the balloon­dilated area was clearly stimulated. In these samples, theDNA synthesis rate was 1.5-fold higher than that of adjacentnonballoon-dilated regions. This increase in DNA synthesiswas consistent with the intimal thickening observed. Auto­radiographs showed a uniform distribution of proliferatingcells with labeled nuclei in the area of intimal thickening.The autoradiographs also demonstrated that the proliferativeactivity shown by the 3H-thymidine incorporation is notsimply due to re-endothelialization, because the proliferatingcells were mostly localized in the intima or media.

DiscussionHistopathology and DNA synthesis. The chronologie,

morphologic and proliferative response of canine renal ar­teries to balloon dilation is similar to the typical response toinjury reported for both angioplasty and other forms of

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Figure 4. Transmission electron micrograph and light micrograph(insert) showed new capillary growth (C) into the media. In addition,proliferative lesions (large arrow, insert) and alterations in theorientations of medial smooth muscle cells (small arrow, insert),suggesting smooth muscle migration and growth, were observed.Bar represents I JLm in transmission micrograph and 30 JLm in lightmicrograph.

endothelial damage in the dog and other animal species(12-17). The early attachment of platelets combined with thelater accumulation of monocytes at the border zones ofdenuded and intact endothelium, and the subsequent releaseof their growth factors, would be expected to have animportant role in the smooth muscle proliferative responsesobserved in our study. This stimulus to smooth musclegrowth may be one component of the occlusion process afterdilation (21-23).

The medial angiogenic response that was also recentlyreported by another group using a canine angioplasty model(24) may also be a component of the occlusion process. Theangiogenic response could be considered a part of the normalprocess of "wound healing" and most likely results from therelease of growth factors from adherent platelets and leuko­cytes. However, an involvement of the angiogenic process inthe development of atherosclerotic plaques in human coro­nary arteries has also been suggested (25). It is unknownwhether the increased number of capillaries observed at thesite of plaque formation is a cause or an effect of the

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Figure 5. Effect of balloon angioplasty on total incorporation of14C-oleate into renal artery lipids. Balloon dilation modified the lipidmetabolic profile of the renal artery. Data at 90 min and 2, 5 and 14days after dilation (angioplasty) represent injured and contiguousnoninjured renal artery. Control data represent nonballoon-dilatedrenal arteries that were divided into segments that reflected anatom­ically the segments from balloon-dilated arteries described as non­injured (hatched bars) and injured (solid bars) (*p < 0.05, **p < 0.01compared with contiguous nonballoon-injured arteries).

atherosclerotic process; however, the presence of thesevessels could certainly provide a means of delivering blood­borne cellular and noncellular mediators to the lesion area.

Arterial lipid metabolism. The observation that angio­plasty produced an increase in steryl ester synthesis (in­creased acyl CoA:cholesterol acyltransferase [ACAT] activ­ity) compared with phospholipid and glyceride synthesis wassomewhat unexpected. Increases in ACAT are usually asso­ciated with increased cellular accumulation of cholesterylester in atherosclerotic blood vessels (13). However, mor­phologic studies of restenotic lesions have generally showncellular proliferation without lipid accumulation (8,9). It maybe that the ACAT response is unrelated to lipid accumula­tion and more related to the proliferative response thatfollows angioplasty. In addition to the stimulation of DNAsynthesis, increased cholesterol synthesis is required for cellproliferation (26). It has been reported that ACAT activityand general cholesterogenesis are increased in proliferatingcells and that cholesterol synthesis appeared to be deregu­lated in these cells (26). That is, increased ACAT activitymay be a part of some permissive process in cell prolifera-

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Figure 6. Upon fractionation of the lipids, it was apparent that thestimulation of lipid synthesis consisted of two distinct components.The first represented a general increase in lipid synthesis. Thesecond was the fivefold increase in cholesteryl ester synthesis, anearly indication of atherogenic changes in the arterial wall. Lipidextracts from the renal arteries described in Figure 5 were fraction­ated into phospholipids (hatched bars), glycerides (diglyceride +triglyceride) (solid bars) and cholesteryl esters (cross-hatched bars).Data shown are the ratios of '4C-oleate incorporated into theballoon-dilated segments (dpm/IOO mg wet weight) to that incorpo­rated into the contiguous nonballoon-dilated segments (dpm/IOO mgwet weight). The control samples represent ratios of anatomicallysimilar sites from arteries not subjected to balloon dilation.

tion that provides adequate lipid for cell membrane synthe­sis. As such, this increase in ACAT activity may playa rolein the gradual reocclusion process that follows angioplasty.

These changes may be accelerated in hyperlipemia,where basal ACAT activity is already elevated (13,14), andmay partially explain the recent observation that moderateelevations in lipoproteins can predict restenosis after angio­plasty (27). These data suggest that additional studies on therole of lipid metabolism in the vascular proliferative re­sponse after angioplasty would be worthwhile.

We appreciate the excellent technical support provided by William R.Humphrey.

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Figure 7. DNA synthesis eH-thymidine accumulation) of balloon­dilated segments was compared with contiguous areas that did notstain with Evans blue dye. Although minimal changes in synthesiswere observed in the first 2 days, by day 5 DNA synthesis was1.5-fold higher in the balloon-dilated segments. This elevation wasmaintained at the 2 week time period. Data shown are the ratios of3H-thymidine incorporated into the balloon-dilated segments com­pared with the contiguous nonballoon-dilated segments (*p ~ 0.05compared with 90 min sample).

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