Focal Toxicity of Oxysterols in Vascular Smooth Muscle ... - Europe PMC

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Mar 28, 1990 - Supported by a gift from Mr. and Mrs. Lev Prichard IlIl of Corpus Christi,. Texas, and by grant HL29680 from the National Institutes of Health. Dr. Guyton is the recipient ..... can block cholesterol synthesis via interaction with an.
American Journal ofPathology, Vol. 13 7, No. 2, August 1990 Copyright © American Association ofPathologists

Focal Toxicity of Oxysterols in Vascular Smooth Muscle Cell Culture A Model of the Atherosclerotic Core Region

John R. Guyton, Brenda L. Black, and Charles L. Seidel From the Departments ofMedicine, Cell Biology, and Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas

Cell necrosis and reactive cellular processes in and near the atherosclerotic core region might result from short-range interactions with toxic lipids. To model these interactions in cell culture, focal crystalline deposits of cholestane-3, 5a, 6jf-triol, 25- OH cholesterol, and cholesterol were overlaid by a collagen gel, on which canine aortic smooth muscle cells were seeded. Oxysterols, but not cholesterol, caused focally decreased plating efficiency and cell death, leading to the formation of a persistent circular gap in the cell culture. Cholestanetriol was largely removedfrom the culture dishes over 3 to 4 weeks, whereas cholesterol and 25-OH cholesterol were largely retained. Smooth muscle cells were motile even in proximity to oxysterol crystals, with occasional suicidal migration toward the crystals. Chemoattraction, however, could not be demonstrated. Despite toxicity, cholestanetriol did not appear to alter the fraction of cells exhibiting 3H-thymidine uptake, even in areas close to the crystals. Thus, oxysterols may be toxic to some cells, without causing major impairment of the migration and proliferation of nearby cells. This would allow the simultaneous occurrence of cell death and proliferation evident in atherosclerosis. (Am J Pathol 1990, 137:425-434)

Most raised lesions or plaques of human atherosclerosis contain a core region characterized by intense lipid deposition and cell necrosis.1-5 From an initial position at some depth within the arterial intima,' the core region spreads or expands with time, undermining the nonthrombogenic luminal surface of the artery.4 Eventually plaque rupture

or ulceration may occur, causing catastrophic thrombotic or thromboembolic events.67 Despite the clinical importance of the core region, the processes that govern its spread or expansion remain largely unknown. Various classes of lipid oxidation products are prime candidates as cytotoxic agents in the core region,8615 and of these, oxysterols are the best character61123 ized at present.8' The pathobiology of the atherosclerotic core region has some unique features, including the fact that a focus of cell necrosis is maintained over long periods in close proximity to relatively normal arterial wall cells capable of migration and proliferation. A hypothesis can be proposed that smooth muscle cells and other arterial wall cells may respond to the presence of a persistent, localized cytotoxic focus in ways that promote atherosclerotic lesion development. It seems profitable to study these responses in a cell culture system, in which a persistent, focal cytotoxic stimulus of limited spatial extent is achieved. This report describes the development of a cell culture model of the atherosclerotic core region, using canine aortic smooth muscle cells grown in proximity to a 2-mm diameter spot of oxysterol crystals-either cholestane-3,3,5a,6f3-triol or 25-OH cholesterol. These oxysterols are autoxidation products of cholesterol known to have toxic effects on vascular smooth muscle.86'0 The oxysterols are sparingly soluble in the culture medium, and their toxic effects are evident, for the most part, only in the immediate vicinity of the crystals. Results on the retention of unaltered oxysterol in the culture dish, cell movement, and cellular DNA synthesis are presented. Despite toxicity to the cells, cell movement and DNA synthesis appear to occur essentially normally in proximity to the oxysterol crystals.

Supported by a gift from Mr. and Mrs. Lev Prichard IlIl of Corpus Christi, Texas, and by grant HL29680 from the National Institutes of Health. Dr. Guyton is the recipient of a Research Career Development Award HL02114 from the National Institutes of Health. Accepted for publication March 28, 1990. Address reprint requests to John R. Guyton, MD, The Methodist Hospital, Department of Medicine A-601, 6565 Fannin, Houston, TX 77030.

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Methods

Smooth Muscle Cell Culture Canine aortas were obtained by sterile technique after euthanasia by an intravenous overdose of sodium pentobarbital (50 mg/kg). Aortas were transported in ice-cold Hank's balanced salt solution (HBSS) without calcium and magnesium. After removal of adventitial fat, aortic segments were incubated in 0.02% collagenase (Type I, Cooper Biomedical, Inc., Malvern, PA) in Dulbecco's modified Eagle's medium (DME, Gibco Laboratories, Grand Island, NY) at 37°C for 20 minutes. The vessel was rinsed with DME and opened longitudinally. Endothelium was scraped away with a modified rubber policeman and discarded. Smooth muscle cells were collected by further scraping and seeded in 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT) in DME with antibiotics added. Within 2 weeks, primary cultures attained confluence and assumed the typical hill-and-valley pattern of smooth muscle cells in culture. They were identified further by electron microscopy showing the presence of myofilaments. Subcultures were seeded at a 1:5 ratio after cell detachment with trypsin and ethylenediaminetetraacetic acid (Gibco). Cells from the third to sixth passage were used for experiments.

Oxysterols Cholestane-3#,5a,6/3-trioI and 25-OH cholesterol were obtained from Steraloids, Inc. (Wilton, NH), and cholesterol from Sigma (St. Louis, MO). Thin-layer chromatography employing hexane:ethyl acetate, 1:2, showed these compounds to be >99% pure. Model of Oxysterol-Cell Interaction For clarity, the final basic procedure for cell culture in the presence of a focal spot of oxysterol crystals is presented here (Figure 1). Several unsuccessful attempts to stabilize crystals mechanically and to provide at the same time a substrate amenable to cell growth are summarized in Results. Sterols were dissolved in ethanol, and 1.2 Ml of solution generally containing 6 Mug total sterols were ejected slowly from a microsyringe to form a spot of approximately 2 mm diameter near the center of a 35-mm culture dish, or on a glass coverslip placed in the dish. Collagen was prepared from rat tails (Pel Freeze Biochemicals, Rogers, AR) and used to form a gel, according to the method of Strom and Michalopoulos.24 This method entailed the mixing of collagen, approximately 1 mg/ml in 0.02 mol/I (molar) ace-

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1.Add sterol in ethanol 2. Dry to form crystals

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gel 4. Dry

5. Add cells Figure 1. Basic procedurefor modeling thefocal interaction of oxysterol crystals with cultured smooth muscle cells.

tic acid, Waymouth's MB 752/1 medium (Gibco), 10-fold concentrated and supplemented with 2% bovine serum albumin, and 0.34 N sodium hydroxide. These components were combined at 40C in volume relations of 17: 2.66:1.10. The mixture was placed over the crystals, 0.7 ml per 35-mm dish, and a gel was obtained by incubating the dishes at room temperature for several minutes. To prevent eventual gel detachment and to increase the proximity of cells to crystals, the dishes were uncovered and allowed to dry overnight in a tissue culture hood. Cells, 25,000 to 60,000 per dish, were seeded into culture dishes prepared in this manner. To facilitate cell attachment, the dishes were incubated overnight with 10% fetal bovine serum in DME before changing to DME with the final serum concentration, which was 2% unless otherwise stated. Media was chang6d twice a week.

Analysis of Sterol Retention After cell culture in the presence of oxysterols or cholesterol for varying periods, duplicate dishes were rinsed, and lipids were extracted with four successive applica-

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tions of 0.25 ml isopropanol. In dishes spotted with 6 gg carrier cholesterol and tracer quantities of 3H-cholesterol, the recovery of label on day 0, before seeding cells, was 73%. Cholesterol losses at this time were ascribed to possible incomplete transfer of cholesterol from microsyringe to the culture dish (a slow process, designed to minimize spot diameter), rehydration of the collagen gel after drying, and possible removal of loose crystals along with the aqueous medium before isopropranol extraction, and extraction inefficiencies. Gas chromatography was used to measure oxysterol retention. Internal standards for gas chromatography were 25-OH cholesterol, when cholestanetriol was the experimental sterol, and vice versa. Sterols were derivatized with N,O-bis(trimethylsilyl)acetamide (Pierce Chemical Co., Rockford, IL) in the presence of 5% trimethylbromosilane (Pfaltz and Bauer, Waterbury, CT). Reaction vials were heated at 90°C for 24 hours, then dried under nitrogen, and the derivatization was repeated once. Vials were not dried after the second derivatization period. Aliquots of 1 to 3 ,ul of the reaction mixture were injected into the gas chromatographic injection port. Chromatography was performed on a Hewlett Packard 571 OA gas chromatograph with flame ionization detector, fitted with a 9foot glass column packed with Gas ChromQ beads coated with 1% SE-30 (Applied Sciences, State College, PA). Column temperature was raised from 180 to 300°C at a rate of 2 '/minute.

Microscopic Techniques Routine microscopic observations were performed by phase microscopy on a Nikon Diaphot. For videomicroscopy, a clear chamber on the microscopic stage was suffused with humidified 95% air/5% C02, and the apparatus was kept at 37°C throughout the period of observation. An Ikegami ITC-47 video camera on the microscope was attached to a Guuyer time-lapse video recorder. Cells were observed at 300:1 increased speed. For electron microscopy, cultures were fixed in 3% glutaraldehyde in 0.13 mol/l Na phosphate buffer and subsequently in 2% osmium tetroxide in HBSS without calcium and magnesium. Dehydration and embedding of the cultures in epoxy resin (Epon, Polysciences, Warrington, PA) was performed in the original culture dish. Thin sections were cut on an LKB Ultrotome Ill, stained with uranyl acetate and alkaline lead citrate, and observed on a JEOL 200CX electron microscope (JEOL USA, Peabody, MA).

Chemotaxis Chemotaxis experiments were performed with blind-well Boyden chambers (Nuclepore, Pleasanton, CA) and poly-

carbonate filters (Nuclepore, 8-,u pore size) coated with gelatin according to Postlethwaite et al.25 Oxysterols were dissolved in appropriate solvents (ethanol for 25-OH cholesterol, dimethylsulfoxide for cholestanetriol) and added in varying concentrations to DME medium with 2% fetal bovine serum. The concentration of lipid solvent in the culture medium was 0.5%; control media contained solvent only. These media, or 100% heat-inactivated serum as a positive control, were placed in the bottom well of the Boyden chamber. In one experiment, crystalline cholesterol, 25-OH cholesterol, or cholestanetriol (2 mg) was placed directly into the bottom well and covered and mixed with DME containing 2% serum (duplicate chambers for each substance). Smooth muscle cells, 60,000 per chamber, were suspended in DME with 2% serum and placed in the upper part of each chamber. Duplicate chambers for each experimental condition were incubated for 6 hours at 37°C in a humidified 5% CO2 atmosphere. At the end of this time, filters were removed, fixed in methanol, and stained with hematoxylin. Cleared, mounted filters were examined at 400X. Cells migrating to the lower filter surface were counted in eight microscopic fields, and results were expressed as a percentage of control.

Tritiated Thymidine Autoradiography The fraction of cells synthesizing deoxyribonucleic acid (DNA) was determined as a function of distance from a 2mm spot of crystals of cholestanetriol or cholesterol. Initial studies used pure cholestanetriol or cholesterol crystals (6 ,ug). Cells were grown in DME with either 1% plateletdeficient equine serum (Hyclone) or the same serum plus 0.5% fetal bovine serum. The platelet-deficient serum and low concentrations of whole blood serum were chosen to limit the overall rate of cell proliferation in the dishes. In a subsequent experiment, cells were grown in 0.7% fetal bovine serum while exposed to crystals formed from mixtures of cholestanetriol and cholesterol. Cells were seeded in 35-mm dishes containing glass coverslips prepared as described earlier. After 3 weeks, cultures were exposed to 3H-thymidine (New England Nuclear, Boston, MA), 6.7 Ci/mmole, at a concentration of 0.5 ,uCi/ml for 12 hours. Cultures were rinsed with phosphate-buffered saline, fixed with neutral buffered formalin, rinsed with water, and air-dried. Coverslips were mounted on glass slides and coated with Kodak NTB-2 autoradiographic emulsion (Eastman Kodak, Rochester NY). After exposure for 5 days, the emulsion was developed according to the manufacturer's guidelines, and subsequently the slides were stained with hematoxylin. Quantitative observations were made on cells located at three randomly selected sites distant from the crystals (about two thirds

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of the distance to the edge of the coverslip) and three sites either overlying the crystals or bordering the zone of cell necrosis. Labeled nuclei were counted per 100X microscopic field, and all nuclei were counted per 400X field.

Results Model Development After formation by solvent evaporation on plastic or glass surfaces, cholesterol and oxysterol crystals were mechanically unstable and tended to detach from the surface on incubation with and changing of tissue culture media. Initial attempts to cover the crystals with agarosegelatin or aldehyde-fixed gelatin treated subsequently with glycine were unsuccessful because of poor attachment of seeded smooth muscle cells. Collagen gels provided good stability to the crystals. Confluent smooth muscle cell cultures sometimes caused detachment of the collagen gel from the edge of the dish, especially when 60-mm dishes were used. Such detachment rarely occurred in 35-mm dishes when the culture technique was modified to include drying and collapse of the collagen gel before adding tissue culture media and cells.

Observations from Phase-contrast Microscopy When smooth muscle cells were seeded onto collagen gels in dishes containing a single spot of cholestanetriol crystals (6 jig in a 2-mm spot), the cells consistently failed to attach to the collagen substrate directly overlying the crystals. This created a clear zone lacking cells over the crystals and extending out 1 to 2 mm from the perimeter of the crystals (Figure 2). The clear zone overlying the crystals was well maintained for 2 to 4 weeks in culture, but after this time cholestanetriol crystals became appreciably attenuated in size, and cell overgrowth occurred. Crystals formed from a mixture of 50% cholestanetriol and 50% cholesterol (3 ,ug of each) induced a somewhat smaller clear zone. Similar crystal spots (total sterol 6 ,ug) containing 5% to 25% cholestanetriol were associated with a zone of variably reduced cell density, whereas crystals containing less than 5% cholestanetriol usually appeared to cause no change in the density of overlying cells. There was little or no attenuation in the size of cholesterol-containing crystals with time; however, the initial cell-depleted zones were at least partially overgrown by 3 to 4 weeks. When smooth muscle cells were seeded in dishes containing a single spot of 25-OH cholesterol crystals (6 ,ug), the cells were able to attach to the collagen substrate

directly overlying the crystals. However, cells throughout the dish were retarded in spreading on the substrate, with decreased spreading evident as late as 7 days after seeding. After 3 weeks, complete absence of cells was observed in a zone overlying the 25-OH cholesterol crystals, and the overall density of cell growth was decreased in the remainder of the dish as well. In dishes containing crystals composed of 10% 25-OH cholesterol and 90% cholesterol, the only appreciable effect was a decrease in cell growth over dense crystal deposits evident 1 to 2

weeks after seeding.

Retention of Cholesterol and Oxysterols As indicated earlier in Methods, the recovery of tritiated cholesterol from dishes spotted with 6 ,ug carrier cholesterol was 73%. The subsequent disappearance of labeled cholesterol from the cell culture system is shown in the top panel of Figure 3. After an initial slight drop between 0 and 3 days, the content of labeled cholesterol was essentially constant in the dish for up to 4 weeks. This result correlated with visual observations of the persistence of cholesterol crystals in the area originally spotted. Data from gas chromatographic measurements of oxysterol persistence in the culture dishes at various times are shown in Figure 3. Dishes were subjected to the application of 6 gg oxysterol (top panel) or 0.6 ,ug oxysterol and 5.4 qg cholesterol (bottom panel) in a single spot. The initial recoveries of oxysterol (time 0) reflect possible losses during formation of the crystalline spot and rehydration of the collagen gel, as well as extraction inefficiencies, which may not have been fully corrected by the use of internal standards. The time courses of disappearance of cholestanetriol and 25-OH cholesterol differed. Cholestanetriol has substantial solubility in aqueous media and thus was removed by changes of the media and possibly by metabolism. The application of crystals of 25-OH cholesterol led to prolonged retention of the oxysterol in the culture dish (top panel of Figure 3). Analogous results were obtained when either cholestanetriol or 25-OH cholesterol was combined with cholesterol in mixed crystals.

Cell Movement and Death Time-lapse observations by videomicroscopy revealed that the motility of cells bordering the clear zone near oxysterol crystals was qualitatively indistinguishable from the motility of cells distant from the crystals or cells grown in control dishes without crystals. The direction of cell migration appeared to be random and unaffected by the presence of oxysterols. On occasion, cells were observed to

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Figure 2. Clear zone, devoid of viable cells, extentding outward from edge of crystalline deposits of cholestanetriol (asterisk). Most cells at the boundary of the clear zone appear healthy. Brightly refractile, roundedforms suggestinig cell death (arrows) are founld in the clearzone. Bar= 100U (XI10).

migrate toward the oxysterol crystals. A close approach to the crystals often resulted in cessation of movement followed by retraction of cell processes, rounding, and blebbing (Figure 4). These observations were consistent with the notion that the clear zone between cells and crystals was maintained by death of cells that approached too close to the toxic crystals. Electron microscopy confirmed the occurrence of cell death in proximity to oxysterol crystals (Figure 5). Swelling of cellular organelles was evident, but a specific mechanism of cell death could not be discerned.

Lack of Chemoattractant Effect The determination of possible chemoattractant effects of oxysterols on smooth muscle cells is summarized in Figure 6. In these experiments, oxysterols were dissolved in dimethylsulfoxide or ethanol before addition to the bottom well of modified Boyden chambers. No significant effects were demonstrated. In addition, macroscopic crystals of oxysterols were tested directly for chemoattractant effect. Migration of smooth muscle cells to the bottom surface of the membrane was never enhanced by oxysterols. In the case of crystalline cholestanetriol, fewer cells were found on both the bottom and top surfaces of the mem-

brane. This result probably was due to cell toxicity and provided no additional information about cell migration.

Effects on DNA Synthesis Proximity to a spot of oxysterol crystals might be hypothesized to cause either a stimulatory or an inhibitory influence on cell proliferation. Cholestanetriol was chosen for these experiments, in preference to 25-OH cholesterol, because the former appeared to have greater focal toxicity for cells-ie, more consistent formation of a clear zone. The results of autoradiographic studies after 3H-thymidine labeling of cells, shown in Table 1, suggest a neutral influence of cholestanetriol on the fraction of cells entering S phase. The thymidine index averaged 9.1% and showed no variation dependent on whether the cells examined were adjacent to cholestanetriol crystals as opposed to distant from the crystals. The same result held true for mixed cholestanetriol/cholesterol or pure cholesterol crystals. The experimental results shown in Table 1 were obtained after maintaining cells for 2 weeks in the presence of 0.7% fetal calf serum. Similar results (not shown) were obtained in the presence of 1.0% plateletdeficient equine serum with or without 0.5% calf serum, and in the presence of fetal calf serum concentrations varying between 0.5% and 4%.

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Formation of Acellular Zone The clear zone, devoid of cells, surrounding spots of oxysterol crystals, appeared to result from a combination of effects. Cells falling within this zone or directly over the

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3 or more weeks, as demonstrated by gas chromatographic analysis. Chromatography gave no evidence for major metabolic conversion of either oxysterol, although this possibility cannot be ruled out. The disappearance of cholestanetriol over 4 weeks may be explained by twice weekly changes of culture medium. The greater retention of 25-OH cholesterol in the culture system is probably due to two factors-lower solubility, leading to persistent crystals, and the tendency for large amounts of 25-OH cholesterol to be incorporated into cell membranes and possibly cytoplasmic sterol esters.16 Crystals also were formed from mixtures of cholesterol and oxysterol. Such mixtures are relevant to the atherosclerotic core region, in which cholesterol remains the predominant sterol constituent.22 When mixtures contained at least 5% oxysterol, a temporary clear zone or a zone of reduced cell density was formed in the cell culture, but the crystals often were overgrown within 3 to 4 weeks.

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Time (weeks) Figure 3. Retentiion of sterols in the cell culture dish. Cholesterol retention ((top panel) was assessed with 3I-cholesterol tracer; oxysterol were measured bj' gas chromatography. The top panelshows rresultsfrom dishes receiving 6,ug ofpure sterol on day 0. The bo ttom panel shows resultsfrom dishes receiving 0. 6,ug of oxysterc)l mixed with 5. 4ug of cholesterol on day zero. Each point showis the mean of measurements in duplicate or triplicate dishes.

Discussion This study demionstrates that a focal toxic site can be incorporated into a cell culture system, resulting in the creation of a persi,stent gap in the culture lasting for at least 4 weeks. Beceause oxysterols are potential agents for causing smooth muscle cell toxicity in atheroscleroSiS,8-11 this cell culture model is particularly applicable to the study of ce,l necrosis and reactive processes in atherosclerosis. if particular interest are the responses of cells bordering the necrotic zone.

Retention of Oxysterols The low aqueotis solubilities of cholestanetriol and 25-OH cholesterol allo wed them to persist in the culture dish for

spreading, suggesting a defect in adherence to the collagen substrate. Thereafter, cells within the zone became necrotic, as suggested by time-lapse videomicroscopy and confirmed by electron microscopy. The necrosis rate appeared to depend on the number of cells migrating

sufficiently close to the crystals to experience a toxic influence, presumably related to a gradient of oxysterol concentration in the culture medium. Toxic effects of oxysterols were first demonstrated in

the 1960s in organ cultures of heart and aorta.8 Subsequently, Taylor and Kandutsch"7 found that oxysterols can block cholesterol synthesis via interaction with an oxysterol-binding protein. They suggested that the consequent depletion of cholesterol (oxysterol applied in serumfree media, hence no lipoprotein cholesterol available) leads to inhibition of cell growth, membrane fragility, and other metabolic effects, but not to decreased viability.1819 Peng and co-workers10 and Naseem and Heald1" used higher concentrations of oxysterol in the presence of serum and observed toxicity to smooth muscle cells. Current understanding of the mechanism of cell toxicity is limited. Suppression of hydroxymethylglutaryl CoA reductase by oxysterols would be expected to inhibit the synthesis of cholesterol and also of other important metabolites such as dolichol, ubiquinone, and the isopentenyladenosine residue of transfer RNA.Y0 A membrane effect of 25-OH cholesterol to increase calcium transport also has been suggested.21

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Figure 4. Videomicroscopy of cell movemenit atnd death in proximity to cholestanetriol crystals (lower left). (a) 0 minute (b) 14i minutes (c) 219 mitnutes (d) 279 minutes. Onte cell (arrow) is observed to migrate toward the boundary of crystal deposits, where it undergoes rounding, gains a refractile appearance, and becomes immobile. Another cell (arrowhead) migrates minimally, then also becomes rounded and refractile. Bar = 50,u (X 190).

Lack of Chemoattractant Effects Because smooth muscle cells were observed to migrate toward oxysterol crystals, despite eventual toxicity to the cells, it seemed appropriate to ask whether a chemoattractant effect similar to that observed with other oxidized lipids26 might be present. Neither cholestanetriol nor 25OH cholesterol had any discernible effect on cell migration in Boyden chambers over a wide range of concentrations. It should be noted, however, that even in the absence of a chemoattractant effect, a net migration of cells from a wound edge into a vacant area can be expected. This phenomenon, observed by videomicroscopy in the present study, is a statistical result of random migration, perhaps aggravated by loss of contact inhibition of cell movement at the wound edge.27

Cell Proliferation An initial hypothesis was that the border zone of the cell layer surrounding the crystals might exhibit an increased

rate of cell proliferation, such as is found at the outer boundary of cell colonies in monolayer culture28 or among cells migrating into the area of a wound created mechanically in similar cultures.'-' Alternatively, the ability of oxysterols in lower concentrations to inhibit cell growth without affecting viability"8 might be expected to decrease the proliferation rate in the border zone. In fact, the observed result was essentially equivalent thymidine indices among cells bordering on and distant from the crystals. It remains to be determined whether this is a balance of opposing effects, or whether the presence of oxysterols simply has minimal effect on cell proliferation under the conditions employed in this study.

Relationship to Wounding Models In vitro wounding of cell cultures, generally performed by mechanical scraping, has proved useful in studies of cell and tissue migration, reactive proliferation, and the main-

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