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J Thorac Cardiovasc Surg 1994;108:556-566
© 1994 Mosby, Inc.

SURGERY FOR ACQUIRED HEART DISEASE

Postoperative reduction of high serum cholesterol concentrations and experimental vein bypass grafts: Effect on the development of intimal hyperplasia and abnormal vasomotor function

Michael L. Klyachkin, MDa, Mark G. Davies, MD, FRCSIa, Jay H. Kim, MDa, Lizzie Barber, BSNa, Helge Dalen, PhDb, Einar Svendsen, MD, PhDa,b, Per-Otto Hagen, PhDa,c


Durham, N.C., and Bergen, Norway

Supported by U.S. Public Health Service grants HL I5448 and TW 04810 and American Heart Association (NC Affiliate) grant NC 92-GB-31. M.G.D. is supported by a National Institutes of Health Fogarty International Research Fellowship (TW 04810) and holds a Royal College of Surgeons in Ireland Surgical Travelling Fellowship and a Trinity College Dublin Postgraduate Scholarship. J.H.K. was supported by the Josiah Charles Trent Memorial Foundation. E.S. was supported by the Norwegian Research Council for Science and the Humanities and the Meltzer's Høyskolefond Foundation.

Presented in part at the Twenty-first World Congress of the International Society for Cardiovascular Surgery, Lisbon, Portugal, September 12-15, 1993.

Received for publication Oct. 1, 1993. Accepted for publication Jan. 9, 1994. Address for reprints: Per-Otto Hagen, PhD, Duke University Medical Center, P.O. Box 3473, Durham, NC 27710.

Abstract

Hypercholesterolemia is an important contributor to the development of intimal hyperplasia and superimposed accelerated atherosclerosis in vein bypass grafts. This study examines the effect of dietary modification of serum cholesterol on the development of intimal hyperplasia and vasomotor function of vein grafts. Thirty male New Zealand White rabbits had a right carotid vein bypass graft and were put to death 28 days after the operation. Twenty animals received a 1% cholesterol diet for 4 weeks before the operation. In 10 animals this diet was continued until harvest (hypercholesterolemia group). In another 10 animals the diet was changed to standard rabbit chow on the day of the surgical procedure and continued until harvest (cholesterol reduction group). The last 10 animals were control subjects. Vein grafts were harvested either for histologic study or for in vitro isometric tension studies. Cumulative dose response curves to norepinephrine, serotonin, bradykinin, and endothelin-1 were determined. After in situ pressure fixation, intimal thicknesses of the vein grafts were measured by videomorphometry. The change in diet produced a 74% reduction in serum cholesterol concentration within 28 days. There was a 26% reduction in the intimal thickness of vein graft intimal hyperplasia and the macroscopic disappearance of atheromatous lesions from the graft wall, which are always observed in vein grafts from the hypercholesterolemia group. Cholesterol reduction did not change hypercholesterolemia-induced agonist supersensitivity. Therefore, cholesterol reduction slows the formation of intimal hyperplasia in vein grafts but does not prevent the persistence of the hypercholesterolemia-associated smooth muscle phenotype. (J THORACCARDIOVASCSURG1994;108:556-66)

Vein bypass grafting remains the most common method of vascular reconstruction to bypass obstructive arterial lesions. Go 1 Intimal hyperplasia develops in these vein grafts after implantation, which results from the proliferation and migration of medial smooth muscle cells into the intima of the graft and the subsequent deposition of connective tissue matrix. Go Go 2-4 Twenty percent of aorta-coronary grafts and up to 50% of peripheral bypass grafts fail within 5 years because of intimal hyperplasia in the short term and the development of atherosclerosis, termed accelerated atherosclerosis, in the long term. Go Go 5-8 Many risk factors for the development of atherosclerosis have been shown to increase the formation of intimal hyperplasia, and reduction of hypercholesterolemia, whether by dietary regulation of cholesterol intake or pharmacologic therapy, increases long-term patency of vein grafts. Go Go 9-12 Experimentally, hypercholesterolemia has been shown to increase both the formation of intimal hyperplasia Go Go 13,14 and the concentration ofcholesterol in vein grafts. Go Go 14,15 Recently, lovostatin treatment and jejunal-ileal bypass surgery have been shown to decrease the cholesterol content of experimental vein grafts, Go 15 although the effect of serum cholesterol reduction on the development of intimal hyperplasia and altered vasomotor function in vein grafts is unknown. The present study shows that reduction of serum cholesterol concentrations decreases the formation of intimal hyperplasia and graft wall lipid accumulation but does not alter vasomotor functional abnormalities induced by cholesterol feeding.

MATERIALS AND METHODS

In 30 male New Zealand White rabbits weighing 2 to 2.5 kg, the ipsilateral external jugular vein was used as an interposition bypass graft to the right carotid artery, as has been described previously. Go 16 Anesthesia was induced and maintained with subcutaneously injected ketamine hydrochloride (60 mg/kg, Ketaset, Bristol Laboratories, Syracuse, N.Y.) and xylazine (6 mg/kg, Anased, Lloyd Laboratories, Shenandoah, Iowa). Twenty animals received a 1% cholesterol diet (ICN Biomedical Inc., Cleveland, Ohio) for 28 days before the operation. In 10 animals, this diet was continued until harvest at 28 days after the operation (hypercholesterolemia group). In another 10 animals, the diet was changed to standard rabbit chow on the day of the procedure, and this new diet was continued until harvest at 28 days. The final 10 animals received standard rabbit chow for the duration of the study. All animals were killed on postoperative day 28 with an overdose of pentobarbital sodium (30 mg/kg intravenously, Anthony Products Co, Arcadia, Calif.). The vein grafts were harvested either for histologic study (n = 4 per group) or for in vitro isometric tension studies (n = 6 per group). Serum cholesterol was determined by the ferric chloride method. Go 17 Animal care complied with the "Principles of Laboratory Animal Care" as formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institutes of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1985).

Histologic study.
After isolation and heparinization (200 IU/kg, intravenously), grafts were perfusion fixed in situ at 80 mm Hg with an initial infusion of Hanks balanced salt solution (HBSS, Gibco Laboratories, Life Technologies Inc., Grand Island, N.Y.) followed by 2% glutaraldehyde made up in cacodylate buffer 0.1 mol/L (pH 7.2) supplemented with sucrose 0.1 mol/L to give an osmolality of approximately 300 mOsm as previously described. Go 16 After fixation, the graft was divided into proximal, middle, and distal thirds from the heel of each anastomosis, and rings for either histologic or functional study were taken from the midpart of the vein grafts where the intimal thickness was uniformly distributed. The histologic specimens were stained with modified Masson's trichrome and Verhoeff's elastin stain. Intimal and medial thicknesses and areas were calculated by videomorphometry (Innovision 150, American Innovision Inc., San Diego, Calif.). An intimal index was also calculated (intimal area/[intimal + medial areas]). Scanning electron microscopy (JEOL JSM-35C scanning electron microscope, JEOL Ltd., Tokyo, Japan) and transmission electron microscopy (Philips 300 transmission electron microscope, N.V. Philips, Eindhoven, The Netherlands) was performed on midportion specimens of the glutaraldehyde-fixed grafts from each group. Go 16

Isometric tension studies.
After isolation and sectioning of a vein graft in situ into 5 mm rings, each vessel ring was suspended from two stainless steel hooks in 5 ml organ baths containing oxygenated Krebs solution (NaCl 122 mmol/L, KCl 4.7 mmol/L, MgCl2 1.2 mmol/L, CaCl2 2.5 mmol/L, NaHCO3 15.4 mmol/L, KH2PO4 1.2 mmol/L, and glucose 5.5 mmol/L at 37° C and bubbled with a mixture of 95% oxygen and 5% carbon dioxide) as previously described. Go 13 The tissues were placed under 0.5 gm tension and allowed to equilibrate in the Krebs solution for 1 hour. During the equilibration period, the solution was replaced every 15 minutes. After equilibration, the resting tension was adjusted in 0.25 gm increments from 0.25 to 2.5 gm and the maximal response to a modified oxygenated Krebs solution containing 60 mmol/L KCl (NaCl 66.7 mmol/L, MgCl2 1.2 mmol/L, CaCl2 2.5 mmol/L, NaHCO3 15.4 mmol/L, KH2PO4 1.2 mmol/L, and glucose 5.5 mmol/L) was measured at each resting tension to obtain a length-tension relationship. On the basis of these results, the optimal resting tension for each ring (the tension at which the response to the modified Krebs solution was maximal) was determined and the ring was set at this tension for subsequent studies. Cumulative dose response curves to norepinephrine (10 -10 to10 -4 mol/L), serotonin (10 -10 to10 -4 mol/L), bradykinin (10 -10 to10 -5 mol/L), and endothelin-1 (10 -12 to10 -7 mol/L) were performed. After precontraction with norepinephrine to give 80% maximal contraction, relaxation in response to sodium nitroprusside (10 -8 to 10 -4 mol/L) was determined. The tissue was allowed to reequilibrate for a minimum of 30 minutes between each experimental run. All drugs were obtained from Sigma Chemical Co. (St Louis, Mo.) except endothelin-1, which was obtained from Calbiochem Co. (San Diego, Calif.). Norepinephrine was initially dissolved in 10 -3 mol/L hydrochloric acid and all other drugs were dissolved in distilled water.

Data and statistical analysis.
The isometric responses of the rings were converted to percent of maximal response and were then plotted against the negative logarithm of the agonist concentration to produce dose response curves. The dose response curves were analyzed and the EC50 values (concentration for the half maximal response) of each ring were calculated by logistic analysis. Go 18 The maximal contractile response of each ring to each agonist is expressed in milligrams of force developed and is then standardized to a contractile ratio (contractile ratio = actual maximal contraction/actual maximal contraction to potassium chloride 60 mmol/L). Data are expressed as the mean ± standard error of the mean with n equal to number of animals. Statistical differences between groups were tested by one-way analysis of variance with Bonferroni correction for multiple comparisons, and a p value less than 0.05 was regarded as significant.

RESULTS

All animals survived until harvest and all grafts were patent on retrieval. Although the serum cholesterol concentration in animals that were returned to a normal diet dropped by 53% at 2 weeks and 74% at 4 weeks, it remained six times higher than that of the normocholesterolemic group (GoTable I). The cholesterol levels in the hypercholesterolemic rabbits did not change significantly over the period of the study (GoTable I).


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  		Table I. Serum cholesterol concentration
 
Gross morphology.
The vein grafts from the hypercholesterolemic animals were surrounded by dense fatty scar tissue. In contrast, the vein grafts from the animals assigned to the cholesterol-reduction diet resembled the vein grafts of the control animals. Areas of intramural accumulations of cholesterol in the middle and distal segments of the grafts were visible in the hypercholesterolemic animals. No such areas were observed in the grafts of the animals that were returned to a normal diet after their operation or in the grafts of the normocholesterolemic animals. Both the hypercholesterolemic and cholesterol-reduction animals had significant increases in vein graft intimal hyperplasia compared with normocholesterolemic animals (Fig. 1, GoTable II). The cross-sectional intimal thicknesses were 83 ± 2 µm, 101 ± 20 µm, and 136 ± 16 µm for the normocholesterolemic, cholesterol-reduction, and hypercholesterolemic groups, respectively (Fig. 2). Although dietary modification resulted in a 26% reduction in the intimal thickness between the cholesterol-reduction and hypercholesterolemic groups, this difference was not statistically significant (p = 0.25).


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  		Table II. Dimensional analysis
 


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  		Fig. 1. A composite photomicrograph of a cross-section of the wall of a hypercholesterolemic graft (A), a cholesterol-reduction graft (B), and a normocholesterolemic graft (C). H, Intimal hyperplasia; M, media. The arrows mark the demarcation between the intimal hyperplasia and the media. (Magnification x100.)

 


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  		Fig. 2. The intimal and medial cross-sectional wall thicknesses of the graft wall of the normocholesterolemic, the hypercholesterolemic, and the cholesterol reduction groups. Values are the mean thickness in micromoles.

 
Ultrastructure.
In scanning electron microscopy, the normocholesterolemic vein grafts had well-preserved endothelial cells with sharp outlines; occasional stomata were visible in the junctional areas of the endothelial cells (Fig. 3, A). In the hypercholesterolemic vein grafts, the endothelial cells were visible but had faint indistinct cellular outlines (Fig. 3, B). There were more frequent stomata and occasional fissures or clefts in the junctions of the endothelial cells in these grafts. In contrast to the hypercholesterolemic vein grafts, the vein grafts in the cholesterol-reversal group were noted to have sharp endothelial cell borders with only occasional stomata in the intercellular junctions (Fig. 3, C).





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  		Fig. 3. A, Representative scanning electron photomicrograph from a normocholesterolemic vein graft showing an intact endothelial cell lining. The endothelial cells have well-preserved, distinct intercellular junctions (arrows). Occasional stomata are noted in the junctional areas (arrowheads). B, Representative scanning electron photomicrograph from a hypercholesterolemic vein graft showing a confluent endothelium. The endothelial cells have visible but somewhat faint cell borders. Some stomata (arrowheads) and fissures or clefts (arrows) can be seen in the junctions between the endothelial cells. C, Representative scanning electron photomicrograph from a cholesterol-reduction vein graft showing a confluent endothelial celllining with sharply defined cell borders and minor stomata in the junctional areas (arrows). (Original magnifications x640.)

 
In transmission electron microscopy, the normocholesterolemic vein graft had an intact endothelial cell monolayer under which were numerous layers of smooth muscle cells representing intimal hyperplasia, and these cells in turn rested on well-organized connective tissue deeper in the wall (Fig. 4, A). Well-defined bundles of peripherally located myofilaments were present in the smooth muscle cells. The intracellular spaces between the smooth muscle cells contained a scattered unorganized electron-dense material that was suggestive of connective tissue. In the hypercholesterolemic vein grafts, transmission electron microscopy showed that the luminal surface was covered by a continuous layer of relatively thick endothelial cells. Beneath this monolayer were multiple layers of smooth muscle cells (Fig. 4, B). Many of the endothelial cells and the smooth muscle cells contained vacuoles. Interspersed between the smooth muscle cells were other cells completely filled with vacuoles (foam cells), which probably represented the final stages of macrophages within the lesion. Active macrophages could be identified by their characteristic extensive slender surface processes (membranes) and by the fact that they contained numerous vacuoles within which was an electron-dense material suggestive of disintegrating products. The number of foam cells and macrophages present varied from region to region. In addition, the intracellular spaces of the subendothelial region were noted to contain an electron-dense material of unknown origin. In the cholesterol-reduction group, the vein grafts had a continuous endothelial cell layer similar to the normocholesterolemic vein grafts in transmission electron microscopy (Fig. 4, C). Unlike the cells of the hypercholesterolemic group, the endothelial cells revealed slender microextensions on their luminal cell surface. The endothelial cells and the smooth muscle cells contained some vacuoles, but the frequency was less than in the hypercholesterolemic vein grafts. In places, the subendothelium contained numerous smooth muscle cells and foam cells. Cells that could be identified as active macrophages were not seen. The intracellular spaces within the intimal hyperplasia were filled with an unorganized ill-defined electron-dense material. In contrast to this material, well-organized connective tissue could be seen deeper in the wall.





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  		Fig. 4. Representative transmission electron photomicrograph of vein grafts from the normocholesterolemic (A), the hypercholesterolemic (B), and the cholesterol-reduction (C), groups. The normocholesterolemic vein graft has an intact endothelial cell monolayer (EC) under which are numerous layers of smooth muscle cells (SMC) representing intimal hyperplasia with well-organized connective tissue deeper in the wall. Well-defined bundles of peripherally located myofilaments are noted in the smooth muscle cells. In the hypercholesterolemic vein grafts, there is a continuous layer of relatively thick endothelial cells, beneath which are multiple layers of smooth muscle cells similar to the normocholesterolemic grafts. However, many of these endothelial cells and the smooth muscle cells contain lipid vacuoles. In addition, interspersed between the smooth muscle cells, there are other cells completely filled with vacuoles (foam cells, asterisk). Active macrophages could also be identified by their characteristic extensive slender surface processes/membranes and by the fact that they contain numerous vacuoles within which are an electron dense material suggestive of disintegrating products. The number of foam cells and macrophages present vary from region to region. In the cholesterol-reduction group, the vein grafts have a continuous endothelial cell layer similar to the normocholesterolemic vein grafts. Unlike the hypercholesterolemic grafts, the endothelial cells reveal slender microextensions on their luminal cell surface. The endothelial cells and the smooth muscle cells contain some vacuoles but the frequency is less than in the hypercholesterolemic vein grafts, and inplaces there are still numerous smooth muscle cells and foam cells in the subendothelium. No active macrophages are seen. (Original magnifications: A, x6000; B, x5500; C x6500.)

 
Vasomotor function.
Vein grafts from hypercholesterolemic animals were more sensitive to serotonin and developed a supersensitivity to both norepinephrine and bradykinin when compared with the control vein grafts (GoTable III). Reduction of serum cholesterol concentrations did not change the increased sensitivity observed in hypercholesterolemia to either serotonin or norepinephrine (GoTable III). A significant decrease in the sensitivity of the vein grafts to bradykinin was observed after cholesterol reduction, approaching the sensitivity of the control grafts to this agonist (GoTable III). Furthermore, the decrease in serum cholesterol concentrations did not produce changes in the response of the vein grafts to endothelin-1. The vein grafts from the cholesterol-reduction group generated absolute maximal contractile forces equivalent to those of the hypercholesterolemic group in response to all agonists with the exception of norepinephrine, which induced a twofold greater maximal contraction in these vein grafts compared with either the hypercholesterolemic or normocholesterolemic vein grafts (GoTable IV). However, when standardized to the potassium chloride responses, the responses in the cholesterol-reduction vein grafts were significantly lower than those in the hypercholesterolemic vein grafts. All grafts responded to sodium nitroprusside in a dose-dependent manner (Fig. 5); however, the grafts from the cholesterol-reduction group had a significantly lower maximal relaxation to sodium nitroprusside (99% ± 1%; p < 0.01) than either the normocholesterolemic (199% ± 51%) or hypercholesterolemic vein grafts (216% ± 29%).


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  		Table III. . Sensitivity
 

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  		Table IV. Maximal contractile force
 


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  		Fig. 5. The maximal responses to norepinephrine, serotonin, bradykinin, and endothelin-1 in vein grafts from normocholesterolemic (n = 6 animals, 12 rings), hypercholesterolemic (n = 6 animals, 12 rings), and cholesterol-reduction (n = 6 animals, 12 rings) groups. Values are the mean ± standard error of the mean of the contractile ratio: *p < 0.05 and **p < 0.01 compared with the normocholesterolemic group and #p < 0.01 compared with the hypercholesterolemic group.

 
DISCUSSION

Schwartz and associates Go 19 have suggested that reduction of both cholesterol and low-density lipoproteins may be useful in either slowing or preventing atherogenesis. They suggest that low-density lipoprotein reduction should be targeted even if the low-density lipoprotein concentrations are within normal limits. In vein grafts, reduction in serum cholesterol concentration by 20% in hypercholesterolemic rabbits with either lovostatin therapy or ileal bypass surgery has resulted in a significant decrease in total graft cholesterol content. Go 15 A reduction of 26% in serum cholesterol concentration at 3.7 years after aorta-coronary bypass surgery in patients who had received combined cholestipol and niacin therapy for 2 years induced regression of atherosclerotic lesions in the native coronary arteries and reduced the occurrence of stenotic and occlusive lesions in the vein bypass grafts of 16% of the patients. These results suggested that reduction of serum cholesterol may improve long-term vein graft patency. Go 11 The results from the present study show that the change in diet from 1% cholesterol to normal diet produced a 74% reduction in serum cholesterol concentrations within 28 days. There was a 26% reduction in the intimal thickness of vein graft intimal hyperplasia and the macroscopic absence of atheromatous lesions in the graft wall. However, the vein grafts in the cholesterol-reduction group were threefold thicker than vein grafts in the normocholesterolemic controls. The fact that postoperative cholesterol reduction did not prevent the increased development of intimal hyperplasia suggests that the elevated serum cholesterol concentrations at the time of vein grafting and shortly thereafter may have been sufficient to induce increased smooth muscle cell proliferation and the increased intimal hyperplasia observed at 28 days. It is conceivable, therefore, that reduction of serum cholesterol in the preoperative period may significantly reduce the formation of intimal hyperplasia. The serum cholesterol concentrations in this study were high and far exceeded those seen in hypercholesterolemic patients. This is in part due to the cholesterol metabolism of the rabbit and in part due to the diet used. However, the higher serum cholesterol concentrations induce rapid development of atheroma in the rabbit and have been used as a model for human atheroma. Thus, although the cholesterol concentrations on a 1% diet are obviously not clinically applicable, the resulting atheromatous lesions are.

The intimal hyperplastic lesions of vein grafts retrieved 1 month after aorta-coronary bypass in human beings have been shown to consist of proliferating smooth muscle cells with only scattered macrophages in the subendothelium, Go 20 and several clinical series have shown the presence of atherosclerotic plaques in long-term vein grafts. Go Go 21,22 The electron micrographs in this study showed that the intimal hyperplastic lesions of the hypercholesterolemic vein grafts were composed predominantly of lipid-laden smooth muscle cells with macrophages in various stages of foam cell formation interspersed between these cells. Cholesterol reduction resulted in a markedly decreased amount of intramural lipid in the smooth muscle cells and the absence of both macrophages and foam cells in the intimal hyperplasia. Similar results have also been reported with cholesterol reduction therapy in the aortas of hypercholesterolemic monkeys. In those studies the results were presumably due to an egress of macrophages from the lesions. Go Go 23,24 The decrease in the quantity of mural lipid and the number of macrophages in the vein graft wall may account for a proportion of the decrease in thickness of the wall observed in the cholesterol-reduction grafts. However, the macrophages is one of the principal cells involved in the development of atherosclerosis through the oxidation of lipoproteins and the formation of lipid peroxides. Go Go 25-27 Oxygen-derived free radicals and lipid peroxides also interfere with the vasomotor function of both endothelial and smooth muscle cells. Go Go 28-30 The decrease in the intramural macrophages, foam cells, and lipids in the vein grafts would therefore decrease the sources and substrates for lipid peroxides and production of oxygen-derived free radicals that may have contributed to both the reduction in wall thickness and the enhanced contractility observed.

Although there was no change in the hypercholesterolemia-induced agonist supersensitivity of the vein grafts to three of four contractile agonists, there was an increase in the absolute maximal contractile force generated by the vein grafts to both receptor-dependent and receptor-independent agonists. Potassium chloride responses reflect calcium fluxes in the tissue, and the global increase in the contractile responses may be a reflection of either increased cell mass due to the formation of intimal hyperplasia or an alteration in calcium-mediated responses of the smooth muscle cells. However, there was a decrease in the thickness of the intimal hyperplasia after the dietary reduction of the serum cholesterol concentrations that would suggest that the probable mechanism for the increased contractility may be altered receptor-independent calcium-mediated responses. Low levels of low-density lipoproteins have been shown to alter calcium metabolism of vascular smooth muscle cell. Go 31 In addition to the enhanced contractility, the ability of the cholesterol-reduction vein grafts to relax was also decreased. The combination of enhanced contractility and poor relaxation in the cholesterol-reduction group suggests that although cholesterol reduction decreases intimal thickness it may increase the severity of any vasospastic event that occurs and also suggests that a nitrodilator may not be as effective in counteracting vein graft spasm. Go Go 32-34

The persistence of the supersensitivity to serotonin in the vein grafts from the cholesterol-reduction group suggests that there has been little change in the receptor characteristics of the smooth muscle cell phenotype present. Enhanced responses to serotonin have been shown to develop early in the aortas of cholesterol-fed rabbits, to precede the development of atherosclerotic lesions, and to increase with the progression of the atherosclerotic disease. Go Go 35-37 However, long-term cholesterol reduction in monkeys with diet-induced atherosclerosis results in the resolution of serotonin hypersensitivity before the anatomic regression of atheromatous lesions. Go 38 This is in contrast to the current study in that the vein grafts showed a decrease in intimal thickness before a significant attenuation of serotonin hypersensitivity occurred. The augmented serotonin response in hypercholesterolemia has been suggested to be due to the direct interaction of oxidized low-density lipoproteins with smooth muscle cells, increased serotonin-activated calcium influx, and/or increased cell-to-cell coupling. Go Go 39-41 The serotonin response in the rabbit vein bypass graft is due to the expression of 5-hydroxytryptamine receptors (type 2), Go 42 and increase in the number or the sensitivity of 5-hydroxytryptamine receptors has been associated with enhanced norepinephrine responses, as was the case in this study. Go 43 It has been shown that increases in norepinephrine sensitivity of rabbit aortic preparations induced by 3 weeks of the dietary cholesterol overload remain unchanged even after 15 weeks of normal diet Go 44; therefore, the increased serotonin and norepinephrine responses in the cholesterol-reduction group of this study indicate that the same functional smooth muscle cell phenotype is expressed in the cholesterol-reduction grafts as in the hypercholesterolemic grafts. These functional changes may be markers for the subsequent development of atherosclerosis, Go Go 37,38 and therefore the vein grafts in the cholesterol-reduction group have retained a phenotypic disposition that may result in the later development of atherosclerosis.

Endothelin-1 is a potent endothelium-derived vasoconstrictive factor and its production appears to be enhanced in atherosclerosis. Go 45 Hypercholesterolemic vein grafts and grafts from cholesterol-reduction animals contracted to endothelin-1 with a greater contractile force than the normocholesterolemic vein grafts. Although the responses to serotonin, norepinephrine, and endothelin-1 were unchanged by dietary reduction therapy, the responses to bradykinin were altered. Whereas hypercholesterolemia substantially increased vein graft bradykinin sensitivity, cholesterol reduction led to an attenuated bradykinin response. Increased sensitivity to bradykinin appears to be characteristic of mature experimental vein grafts when compared with unmanipulated veins. Go 46 The role and the mechanism whereby the alterations in the sensitivity and contractile force induced by bradykinin occur are still unclear. Changes in bradykinin receptor subtypes and an increased degradation of bradykinin by increased local concentrations of angiotensin-converting enzyme may be responsible for these changes. Go Go 46,47

In conclusion, postoperative cholesterol-reduction therapy results in the decreased formation of vein graft intimal hyperplasia without significant attenuation of the hypercholesterolemia-induced augmentation in smooth muscle cell contractile responses. Cholesterol reduction thus slows the morphologic changes in vein grafts but does not alter the changes in vein grafts smooth muscle cell phenotype induced by hypercholesterolemia.

Acknowledgments

The technical assistance of Mrs. Anne-Marie Sandsbakk-Aüstarheim and Mrs. Turid Foldnes-Gülbrandsen is greatly appreciated. Microsutures were a gift of Ethicon, Inc., Somerville, New Jersey.

Footnotes

From the Vascular Biology and Atherosclerosis Research Laboratory, Departments of Surgery and Biochemistry, Duke University Medical Center, Durham, N.C., and Department of Pathology, The Gade Institute, University of Bergen, Bergen, Norway. Back

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