Nifedipine reduces atherogenesis in cholesterol-fed heterozygous WHHL rabbits

195

Atherosclerosis, 84 (1990) 195-201 Elsevier Scientific Publishers Ireland. Ltd.

ATHERO

04536

Nifedipine reduces atherogenesis in cholesterol-fed heterozygous WHHL rabbits James B. Atkinson and Larry L. Swift Department

of Pathology, Vanderbilt University and Nashville Veterans Administration

Medical Center, Nashville, TN (U.S.A.)

(Received 26 March, 1990) (Revised, received 15 May, 1990) (Accepted 18 June, 1990)

We have developed a new model to study the interaction between diet and genetics in atherogenesis, the cholesterol-fed heterozygous WHHL rabbit. To determine the effects of calcium blockers on atherosclerosis in this model, two groups of heterozygous WHHL rabbits were fed 0.25% cholesterol and 2% peanut oil with (n = 6) and without (n = 6) oral nifedipine (40 mg/ kg/ day) for 16 weeks. Body weights, serum cholesterol, triglycerides and calcium, and blood pressures were not significantly different between the 2 groups during the study period. Heterozygous WHHL rabbits in the nifedipine group had less aortic surface area with sudanophilic lesions (23 f 15% vs. 62 + 18% P c:0.01) and fewer segments of coronary arteries with lesions (19 f 9% vs. 35 f 88, P < 0.02). Total aortic cholesterol, phospholipid, and calcium were also reduced in nifedipine-treated rabbits compared with untreated animals. We conclude that nifedipine reduced atherosclerosis in this model. Although the mechanism is unknown, it is apparent that nifedipine acts independently of changes in plasma lipids and blood pressure.

Key words: Atherosclerosis;

Calcium channel blockers; Hypercholesterolemia;

Mmduction

Calcium channel blockers, therapeutic agents used for treatment of hypertension and angina pectoris [I], have been reported to reduce the severity of experimental atherosclerosis [2]. Several publications have described effects of calcium

Correspondence to: James B. Atkinson, M.D., Ph.D., Department of Pathology, Vanderbilt University, Nashville, TN 37232, U.S.A.

0021-9150/9o/sO3.50

6 1990 Elsewier scientific

Publishers

WHHL rabbits

channel blockers on atherosclerosis in hypercholesterolemic rabbits [3-181. Among the various calcium blockers studied, nifedipine has been the most effective in reducing atherosclerosis. However, these studies have utilized the cholesterol-fed rabbit, and atherosclerotic lesions in this model can be quite different from those seen in humans. In fact, the lesions produced in the cholesterol-fed rabbit may resemble those observed in lipid storage .disorders more than in human atherosclerosis P91.

Ireland, Ltd.

196

Homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, like patients with familial hypercholesterolemia, have a marked hypercholesterolemia resulting from a deficiency of functional low density lipoprotein (LDL) receptors [20]. They develop atherosclerotic lesions similar in many respects to those found in humans with advanced atherosclerosis [21,22]. Using the homozygous WHHL rabbit model, however, neither nifedipine nor verapamil has been effective in reducing the extent or severity of atherosclerosis [7,11,15], suggesting that they interact through the LDL receptor. Work in our laboratory has been directed toward characterizing cholesterol-fed heterozygous WHHL rabbits as an animal model to study the interaction between diet and genetics in atherosclerosis [23]. We showed that heterozygous WHHL rabbits fed cholesterol for up to 24 weeks developed complex lesions in the aorta and coronary arteries, with necrosis, cholesterol clefts, fibrous caps and calcification [23]. These lesions were different from those found in cholesterol-fed New Zealand white (NZW) rabbits, in which foam cell-rich lesions were observed. Extravascular lipid deposits were also less extensive than in control rabbits fed comparable diets [23]. Based on these results, we suggested that the cholesterol-fed heterozygous WHHL rabbit may mimic a larger segment of the population who have a genetic predisposition towards atherosclerosis and, in addition, consume a diet rich in fat and cholesterol [23]. We therefore undertook a study to determine the effects of nifedipine on atherogenesis using this new animal model. Materials and methods Study protocol Our colony of WHHL

rabbits was established from a breeding pair provided by Drs. Joseph L. Goldstein and M.S. Brown, Dallas, TX. Heterozygous rabbits were obtained by breeding male homozygous WHHL rabbits to NZW females. For this study, 12 male WHHL heterozygous rabbits, 2 months of age, were randomized to a nifedipine group and a control group. Rabbits were fed 0.25% cholesterol diets prepared by dissolving cholesterol (Nutritional Bio-

chemicals, Cleveland, OH) in hot peanut oil (Planters, Nabisco Brands, East Hanover, NJ), and mixing with Teklad rabbit diet (containing 2% fat) (Teklad, Madison, WI) at a final concentration of 0.25% cholesterol and 2% peanut oil (w/w). For the nifedipine diets, nifedipine (Sigma Chemical Company, St. Louis, MO) was dissolved in ether, mixed into the cholesterol-enriched diets for a final concentration of 40 mg nifedipine per 50 g food, and placed in open containers at 4°C in the dark to allow the ether to evaporate. The control diets were prepared in an identical manner, except nifedipine was omitted. All rabbits were fed 125 g of food per day, and the daily dosage of nifedipine was 40 mg/kg/day. These diets were prepared fresh weekly and stored at 4°C in the dark. Rabbits were weighed weekly. Arterial blood pressures were recorded at the beginning of the study and at 4-week intervals using a Gould P23Db pressure transducer and Gould RS 3200 amplifier and recorder (Gould Electronics, Cleveland, OH), as described by Henry and Bentley [3]. Blood pressure measurements were recorded 20 min after placement of the transducer catheter to minimize the effects of stress, and rabbits did not appear visibly agitated during the recordings. Blood was collected from an ear vein every 4 weeks for plasma lipids (total cholesterol and triglycerides), calcium, and nifedipine analyses. Total serum cholesterol and triglycerides were analyzed enzymatically using diagnostic kits (Sigma), and serum calcium was assayed by use of ocresolphthalein complex (Sigma) [24]. For serum nifedipine analysis, blood was collected at consistent times during the day (but no attempt was made to alter dietary intake) and a modified radioreceptor assay as described by Gould et al. [25] was performed. At the end of 16 weeks, rabbits were anesthetized with Nembutal(40 mg/kg body weight; Abbott Laboratories, North Chicago, IL), a midline incision was made, and the animals were exsanguinated from the abdominal aorta. Morphologic

analyses

At the time of death, the heart and aorta were removed en bloc, and the aorta was isolated at the origin from the aortic valve to 1 cm beyond the bifurcation of the common iliac arteries. Adventitia was dissected away and the aorta was opened

197 longitudinally along the mid-posterior wall. It was photographed endothelial side up and then divided into two equal longitudinal halves; the left half was pinned endothelial side up and placed in 2% paraformaldehyde, pH 7.4. The right half was frozen and stored at - 70°C for biochemical analysis. After overnight fixation, the fixed specimen was stained with Sudan IV [26] and again photographed. The extent of sudanophilia was determined by planimetry using a Beseler projector Dichro 67 and a HIPAD digitizer (Houston Instruments, Austin, TX) interfaced with an IBM-XT computer. For light microscopy, sections of the fixed aorta were obtained from 2 standard sampling sites (immediately distal to the branch of the left subclavian artery for a distance of 2 cm, and 2 cm immediately proximal to the bifurcation). They were processed, embedded in paraffin, and 4-pm sections were cut and stained with hematoxylin and eosin (H&E) and Movat pentachrome stains [27]. Maximal intimal thickness in each section was measured using a calibrated reticule; the media immediately beneath the area of maximal intimal thickness was also measured. The left and right coronary arteries of the excised heart were ‘perfused through the root of the aorta at 100 mm Hg with a mixture containing 2% paraformaldehyde, pH 7.4, and barium-gelatin pigment (to facilitate identification). The hearts were immersed in fixative for 24 h, and the major coronary arteries (left main, left anterior descending, left circumflex, and right coronary arteries) were removed en bloc, sectioned at l-mm intervals, processed for light microscopy, and stained with H&E and Movat pentachrome stains. Slices of myocardium were made parallel to the posterior atrioventricular sulcus at 2-mm intervals, and whole-mounted sections were processed for light microscopy and stained with H&E and Masson trichrome stains. The presence and absence of lesions within each coronary artery segment examined microscopically was recorded.

streaked on TLC plates (precoated Silica gel 60, EM Laboratories, Elsmford, NY). The plates were developed in petroleum ether/diethyl ether/acetic acid (80 : 20 : I), stained with 0.05% Rhodamine B, and the individual lipids were identified by comparison with a standard lipid mixture (Non-polar Lipid Mix A, Supelco, Inc., Bellefonte, PA). Unesterified cholesterol, cholesterol esters, and phospholipids were eluted from the silica gel as described previously [29]. Cholesterol was determined by the method of Babson et al. [30]. Total lipid phosphorous in the phospholipid fraction was determined by the method of Bartlett [31], using a factor of 25 to convert lipid phosphorous to phospholipid. Proteins were estimated by the method of Lowry et al. [32]. Data are expressed as mean f SD, and Student’s f-test was used to determine statistical significance. All analyses were made blinded with regard to the control versus nifedipine group.

Biochemical

Morphologic

analyses

Lipids were extracted from the frozen aorta samples by the method of Folch et al. [28] and separated using thin-layer chromatography (TLC) techniques. Lipids (4-8 mg total lipid) were

Results

All rabbits gained weight steadily and remained healthy during the 16 weeks of the study. No significant differences in mean arterial blood pressure were found, with pressures at the end of the study 85.3 f 2.3 mm Hg for the control group and 83.2 + 1.8 mm Hg for the nifedipine group. All nifedipine-treated rabbits had comparable serum drug levels at all times studied, and the average level at the time of sacrifice was 685 -J 120 ng/ml (Fig. 1). Serum cholesterol and triglyceride levels at the beginning and end of the study are shown in Table 1. Addition of 0.25% cholesterol to the diet resulted in elevation of serum cholesterol to the 600-700 mg/dl range, and no differences were apparent when comparing control and nifedipinetreated WHHL heterozygous rabbits. Although serum calcium was slightly lower in the nifedipine group at the end of the study (Table l), the differences did not reach statistical significance. analyses

Aortic surface tive lesions after shown in Table had significantly

area involved by Sudan IV-posi16 weeks of cholesterol feeding is 2. The nifedipinatreated group less sudanophilia as compared to

198 eoor

-sL

2

-+iWEEKS

Fig. 1. Serum nifedipine levels in cholesterol-fed heterozygous WHHL rabbits. TABLE 1 SERUM LIPID AND CALCIUM LEVELS OF HETEROZYGOUS WHHL RABBITS FED 0.25% CHOLESTEROL FOR 16 WEEKS, WITH AND WITHOUT NIFEDIPINE TREATMENT Values are expressed as mean f SD (mg/dl). Triglycerides

Calcium

Control (n = 6) Start 88* 25 End 758 f 126

103f24 183f27

13.0*0.5 12.8kO.4

Nifedipine (n = 6) 74* 21 Start End 668k117

8Ok20 226 f 88

12.3f0.5 11.9f0.6

Total cholesterol

TABLE 2 PERCENT OF AORTA INVOLVED BY SUDANOPHILIC LESIONS, MEAN INTIMA/MEDIA THICKNESS RATIO OF AORTA, AND PERCENTAGE OF CORONARY ARTERY SEGMENTS WITH LESIONS (91 CA LESIONS) IN CONTROL AND NIFEDIPINE-TREATED CHOLESTEROL-FED HETEROZYGOUS WHHL RAE BITS Nifedinine

Control

(n=6)

(n==6)

P value

% Sudanophilia

22.6 i14.7

62.2 kl8.3

< 0.01

Intima/media ratio Thoracic aorta Abdominal aorta %CA lesions

0.49* 0.19 0.26f 0.20 18.5 f 9.2

0.92* 0.38 0.85* 0.36 35.0 f 7.6

< 0.05 < 0.05 < 0.02

Fig. 2. Sudan IV-stained aortas from cholesterol-fed WHHL heterozygous rabbits in the nifedipine (A) and control (B) groups. This pair was studied and killed simultaneously. Raised lesions were only seen in the thoracic aorta and around small branches of the abdominal portion of the aorta from the nifedipine-treated rabbits (arrows). In contrast, virtually the entire aorta was involved by raised, sudanophilic lesions in the control rabbit, with only focal areas of normal intima (arrowheads).

the control group. The thoracic aorta developed lesions earlier than did the abdominal aorta, a finding that appears to be universal in rabbit models, but both portions were near-equally involved in the control group of rabbits as disease progressed (Fig. 2). The abdominal aorta had less sudanophilia than the thoracic portion in the nifedipine-treated group (Fig. 2). Sections obtained at standard sampling sites in both the thoracic and abdominal aorta of cholesterol-fed WHHL heterozygous rabbits in the control group had thicker intimas compared to nifedipine-treated rabbits, as indicated by larger intima to media ratios (Table 2). Standard deviations were large, owing to variability in location of lesions at the sampling sites, but the differences were still statistically significant. Approximately one-third of coronary artery segments from WHHL heterozygous rabbits in the control group had microscopic evidence of atherosclerotic lesions, compared to just under 19% of those segments examined in the nifedipine group (Table 2, P c 0.02). The vast majority (72%) of coronary segments that contained atherosclerotic lesions had less than 50% reduction in crosssectional area, and segments with total occlusion were only rarely observed.

199 TABLE 3 COMPOSITION OF AORTA FROM CONTROL AND NIFEDIPINE-TREATED CHOLESTEROL-FED HETEROZYGOUS WHHL RABBITS Values are expressed as mean + SD.

Total cholesterol a Cholesterol ester ’ Phospholipid ’ Calcium b

Nifedipine (n=6)

Control (n=6)

P value

3.7*1.3 1.9*0.4 6.9k5.0 2.6kl.l

6.5 rt 2.1 4.3 + 1.1 14.7k4.4

< < < <

4.3*1.3

0.05 0.001 0.02 0.05

’ Expressed as mg/gm wet weight. b Expressed as mg/gm dry weight.

Biochemical studies Table 3 shows results of biochemical analyses of aortas from cholesterol-fed WHHL heterozygous rabbits in the control group compared to those that received nifedipine. Mean values for aortic total cholesterol, cholesterol esters, and phospholipids were significantly lower in the nifedipine-treated group compared to the nontreated group. Aortas from nifedipine-treated WHHL heterozygotes also had less calcium compared to those from the control group (Table 3). Discussion

The antiatherogenic potential of calcium channel blockers in experimental animal models has been the subject of numerous published studies (see refs. 2 and 33 for review). Although some results are conflicting, probably due to different agents and different animal models, most of the data suggest that these drugs may reduce atherosclerosis. Preliminary data from ongoing clinical trials in humans also indicate possible beneficial effects of nifedipine and nicardipine, particularly in early (minimal) coronary artery lesions, but not in advanced disease [34,35]. A notable exception in rabbit models has been the inability of calcium channel blockers to reduce atherosclerosis in homozygous WHHL rabbits [7,11,15]. This suggests that interaction between calcium blockers and the LDL receptor is responsible, at least in part, for the antiatherogenic effects of these agents.

Some mechanisms by which calcium blockers might reduce atherosclerosis include LDL receptor-mediated inhibition of 3-hydroxy-3-methylglutaryl coenzyme A activity [36], stimulation of LDL receptor synthesis [37], and inhibition of degradation of internalized LDL [38]. In addition, these agents may act through numerous other processes related to endothelial function, smooth muscle cell migration and proliferation, and synthesis of extracellular matrix components [33]. Verapamil has been shown to increase uptake and degradation of LDL by cultured human skin fibroblasts obtained from normal controls, but failed to do so in fibroblasts from patients with homozygous familial hypercholesterolemia [39]. Verapamil also enhanced binding and intemalization of LDL in cultured human monocyte-derived macrophages [40]. It has also become apparent that these agents do not have the same effects; Paoletti and colleagues have reported that verapamil and diltiazem are effective in modulating receptor-mediated LDL catabolism in vitro, whereas dihydropyridines (except amlodipine and flunarazine) are inactive [41]. The cholesterol-fed heterozygous WHHL rabbit offers an animal model to study the interaction of diet and genetic propensity for atherogenesis. It also has the potential to answer some of the questions concerning the role of the LDL receptor and LDL receptor-mediated functions in suppression of atherogenesis by calcium channel blockers. Using this model, we observed that 40 mg nifedipine per kg per day for 16 weeks reduced the extent of atherosclerosis in the aorta and coronary arteries. This was accompanied by a reduction in aortic lipids and calcium, but with no apparent changes in blood lipids or calcium levels, or blood pressures. Aortas from nifedipine-treated rabbits in our study had decreased cholesterol and phospholipids compared with the control rabbits, similar to the findings of others [15]. Although one might expect cholesterol and phospholipid to exhibit an inverse relationship, our findings might be explained by the fact that an aortic media-intima sample was analyzed, and the untreated rabbits had marked increases in raised, lipid-containing lesions compared to the nifedipine-treated group.

200

While the dosage of nifedipine exceeded that which is used clinically in humans, rabbits metabolize calcium channel blockers differently; rabbits have a high first-pass metabolism in the liver for many of these agents, and oral dosages 5-10 times higher than those required in humans are necessary to obtain similar plasma concentrations [12]. We still recognize the need to perform similar experiments using smaller doses of nifedipine, however, before these data can be considered directly applicable to humans. Alternatively, calcium channel blockers which have longer halflives, or which have the capacity to bind to receptors for longer periods before dissociation and/or degradation, may prove to be beneficial. For example, amlodipine, a second-generation dihydropyridine derivative which has a slow onset and longer duration of action than nifedipine, has been reported to reduce atheroma formation when given at a dosage of 1 mg/kg/day to rabbits fed 0.1-2X cholesterol for 8 weeks [42]. The onset of treatment may have bearing on the success or failure of calcium channel blockers in retarding atherosclerosis. In animal models, calcium antagonists have little effect if rabbits are fed cholesterol for greater than 8 weeks before the onset of drug therapy [33,43], and preliminary human data support the concept that these agents are ineffective on established, advanced plaques [34]. Our findings in the cholesterol-fed WHHL heterozygous rabbit model support those observations; initiation of nifedipine treatment concurrently with cholesterol feeding reduced the extent of development of atherosclerosis, and this was associated with less aortic calcium. We did not observe any of the advanced lesions in the nifedipine-treated group, with calcification and necrosis, that are characteristic for this model [23], although this study did not specifically quantitate lesion composition. Regardless of whether animal models are completely applicable to the human situation, the use of calcium channel blockers may potentially unravel the mechanisms of atherogenesis as they relate to the numerous cellular processes influenced by calcium. Future studies in our laboratory will address several LDL receptor-dependent and -independent mechanisms in the cholesterolfed heterozygous WHHL rabbit model that may

have a bearing on the relationship between calcium and the development of atherosclerosis. Acknowledgements

The authors acknowledge the excellent technical assistance provided by Ms. Linda Gleaves and Mr. Man& Paul. This work was supported by research funds from the Veterans Administration and in part by NIH DK 26652. It was presented at the American Heart Association 62nd Scientific Sessions, New Orleans, LA, November 1989. References 1 Gpie, L.H., Calcium antagonists, Lancet, 1 (1980) 806. 2 Kjeldsen, K. and Stender, S., Calcium antagonists and experimental atherosclerosis, Proc. Sot. Exp. Biol. Med., 190 (1989) 219. 3 Henry, P.D. and Bentley, K.S., Suppression of atherogenesis in cholesterol-fed rabbit treated with nifedipine, J. Clin. Invest., 68 (1981) 1366. 4 Ginsburg, R., Favis, K., Bristow, M.R. et al., Calcium antagonists suppress atherogenesis in aorta but not in intramural coronary arteries of cholesterol-fed rabbits, Lab. Invest., 49 (1983) 154. 5 Rouleau, J-L., Parmley, W.W., Stevens, J. et al., Verapamil suppresses atherosclerosis in cholesterol-fed rabbits, J. Am. COB. Cardiol., 6 (1983) 1453. 6 Stender, S., Stender, I., Nordestgaard, B. and Kjeldsen, K., No effect of nifedipine on atherogenesis in cholesterol-fed rabbits, Arteriosclerosis, 4 (1984) 389. 7 Van Niekerk, J.L.M., Hendriks, T., De Boer, H.H.M. and Van ‘t Laar, A., Does nifedipine suppress atherogenesis in WHHL rabbits?, Atherosclerosis, 53 (1984) 91. 8 Naito, M., Kuzuya, F., Asai, K. et al., Ineffectiveness of Ca’+-antagonists nicardipine and diltiazem on experimental atherosclerosis in cholesterol-fed rabbits, Atherosclerosis, 51 (1984) 343. 9 Blumlein, S.L., Sievers, R., Kidd, P. and Parmley, W.W., Mechanism of protection from atherosclerosis by verapamil in the cholesterol-fed rabbit, Am. J. Cardiol., 54 (1984) 884. 10 Willis, A.L., Nagel, B., Churchill, V. et al., Antiatherosclerotic effects of nicardipine and nifedipine in cholesterol-fed rabbits, Arteriosclerosis, 5 (1985) 250. 11 Tilton, G.D., Buja, M., Bilheimer, D.W. et al., Failure of a slow channel antagonist, verapamil, to retard atherosclerosis in the Watanabe heritable hyperlipidemic rabbit: an animal model of familial hypercholesterolemia, J. Am. Coll. Cardiol., 6 (1985) 141. 12 Stender, S., Ravin, H., Haugegaard, M. and Kjeldsen, K., Effect of verapamil on accumulation of free and esterified cholesterol in the thoracic aorta of cholesterol-fed rabbits, Atherosclerosis, 61 (1986) 15.

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