Dissociation Between the Antiatherosclerotic Effect of Trandolapril and Suppression of Serum and Aortic Angiotensin-Converting Enzyme Activity in the Watanabe Heritable Hyperlipidemic Rabbit
Abstract This study was undertaken to determine whether low doses of the angiotensin-converting enzyme (ACE) inhibitor trandolapril affected atherosclerosis in the Watanabe heritable hyperlipidemic (WHHL) rabbit. Trandolapril (10 μg/kg body weight per 48 hours) was begun at 3 months of age and continued for 9 months. The selected dose reduced serum ACE activity but did not influence blood pressure. Both serum and aortic ACE activity were reduced by more than 80% in the trandolapril-treated compared with control WHHL rabbits, similar to the suppression achieved with the 25-fold-higher dose that in our previous studies induced marked inhibition of aortic atherosclerotic lesions in the WHHL rabbit. In contrast to these prior findings, low-dose trandolapril had no effect on aortic surface involvement by atherosclerosis, aortic cholesterol content, or aortic morphology. The data suggest that the antiatherosclerotic action of ACE inhibitors in the WHHL rabbit may not be related directly to arterial enzyme inhibition.
- angiotensin-converting enzyme inhibitors
- blood pressure
- angiotensin-converting enzyme
Several studies demonstrated that angiotensin-converting enzyme (ACE) inhibitors can hinder the development of atherosclerosis in hypercholesterolemic normotensive animals. The effect has been observed with different ACE inhibitors and across a wide range of species, including the Watanabe heritable hyperlipidemic (WHHL) rabbit,1 2 cholesterol-fed rabbit,3 monkey,4 minipig,5 and hamster.6 Various actions of these drugs have been proposed as contributing to antiatherosclerotic effects such as inhibition of growth or migration of arterial smooth muscle cells by reducing angiotensin II or increasing bradykinin and prostacyclin levels, improvement of endothelium-dependent function, inhibition of leukocyte adherence and/or penetration into the arterial intima, inhibition of oxidation of lipoproteins, and lowering of blood pressure.2 3 6 7 8
In our prior studies in the normotensive WHHL rabbit, we observed marked inhibition of aortic atherosclerosis by captopril and trandolapril in association with a reduction in cellularity of atherosclerotic plaques.1 2 The blood pressure of the animals also was reduced with decreases in systolic blood pressure of 10 to 20 mm Hg observed in response to both ACE inhibitors. In addition, thickness of the arterial media of the treated WHHL rabbits was much less than that of control rabbits, suggesting a response to the lowering of blood pressure. Because these data were provided in prior publications, they are not repeated here. However, they were used for comparisons with the new findings.
The current investigation was undertaken to determine whether the antiatherosclerotic effects of ACE inhibitors could be dissociated from their blood pressure lowering or arterial ACE-inhibiting actions. WHHL rabbits were treated with low doses of trandolapril that were insufficient to reduce blood pressure but still adequate to inhibit serum and aortic ACE activity markedly. We examined the development of atherosclerosis in these rabbits by using our previously established protocols.1
This study used 16 WHHL rabbits bred in our core facility. After the rabbits were weaned at 2 months of age, they were fed 120 g/d Agway Prolab High Fiber Rabbit Chow. The rabbits were housed separately and had unlimited access to water. Their maintenance was essentially identical to that described previously.1
The rabbits were assigned randomly to control or drug treatment groups at 3 months of age. Treatment consisted of trandolapril 10 μg/kg every other day. This dose was selected because it had no significant effect on blood pressure but still caused a significant reduction of serum ACE activity. To determine this dose, preliminary titration studies in separate animals were performed with trandolapril 2.5, 5, 10, 25, 100, and 250 μg/kg for 1- to 2-week periods each before blood pressure and serum ACE measurements were made. Because of the long duration of the effect of trandolapril, the drug was administered only on alternate days, as in our prior study. The control group was treated identically to the trandolapril group, except the drug was omitted.
Systolic blood pressure and heart rate were measured at monthly intervals, as previously described.1 9 In brief, unanesthetized rabbits were placed into a Styrofoam restraining box, their tails were shaved, and a tail cuff with a photoelectric cell detector was used to determine systolic pressure.
Blood was obtained from the central ear artery after an overnight fast for assay of serum cholesterol concentrations.1
To determine whether aortic and serum ACE activity was inhibited by low- and high-dose treatment with trandolapril, ACE measurements were performed in four drug-treated and three control WHHL rabbits at 2 or 4 weeks after the trandolapril treatment was begun. For the aortic assays, the rabbit was anesthetized with pentobarbital, the thoracic aorta was removed and rapidly stripped free of adventitia, and the tissue was frozen at −80°C. ACE activity was measured according to our modifications10 of the method of Cheung and Cushman11 by use of 14C-labeled Hip-His-Leu (purchased from Dupont NEN Research Products) as substrate. The aorta was quickly homogenized in 1.0 mL medium containing 100 mmol/L potassium phosphate buffer (pH 8.3) and 300 mmol/L NaCl. The homogenate was centrifuged at 1000g for 10 minutes, and duplicate aliquots of the supernatant were used for enzymatic analysis and protein determination.12 Aortic extracts or serum samples were added to an incubation mixture containing 100 mmol/L potassium phosphate buffer (pH 8.3), 300 mmol/L NaCl, 2.5 mmol/L Hip-His-Leu, and 14C-labeled Hip-His-Leu (0.83 μCi/mL buffer) in a final volume of 0.24 mL. After incubation at 37°C for 4 hours (aorta) or 1 hour (plasma), 100 mL of 2.5 mol/L HCl was added; then labeled hippuric acid was extracted with ethyl acetate, and its radioactivity was determined.
Throughout the 9-month study period, serum ACE also was assayed in selected animals and in the group that had aortic ACE measured.
At the completion of the study, the rabbits were killed with pentobarbital (100 mg/kg IV). The body was perfused for approximately 20 minutes with 10% acetate-buffered formalin at the mean blood pressure of the animal. The aorta was removed, cleaned free of adventitia, and fixed in formalin. To determine the extent of the aortic involvement by atherosclerosis, the aorta was opened longitudinally to expose the intimal surface and pinned onto black wax without stretching, as described.1 Photographs were taken and projected onto a 12×12-in. digitizing tablet (model SD 420E, Wacom Technology, Inc), and the total area and lesion areas were traced. The National Institutes of Health image 1.49 processing and analysis program for the Macintosh computer was used to calculate the areas.
After the aortic specimens were photographed, they were divided into ascending aorta plus arch, descending thoracic aorta, and abdominal aorta. Free and ester cholesterol contents were assayed as previously described.1
Representative segments of descending thoracic aorta were removed for microscopic evaluation. The sections were stained with hematoxylin and eosin before examination. Medial thickness was measured with a Javelin video camera (model JE 7362) with a Sony Trinitron monitor and optimas 402 software.
Statistical analyses of data on body weight, serum cholesterol, heart rate, and blood pressure were performed by two-way ANOVA with corrections for repeated measures with sas (SAS Institute Inc.). Aortic cholesterol levels were analyzed with Student’s t test for independent measures. The data on aortic surface involvement by atherosclerosis were compared by the Wilcoxon rank-sum test.
In the titration studies used to determine the trandolapril dose for the long-term protocol, both 25- and 100-μg/kg alternate-day dosing produced some lowering of blood pressure, whereas the 2.5-, 5-, and 10-μg/kg doses did not. The 10-μg/kg amount was the lowest dose that reduced serum ACE consistently; therefore, it was chosen for the long-term protocol.
All rabbits appeared to be in good health throughout the study. Those treated with trandolapril tolerated the drug without apparent adverse effects.
Body weight, serum cholesterol, and heart rate were comparable in control and trandolapril-treated rabbits at the beginning of the study. Body weight increased at a comparable rate in the two groups during the 9-month study (Fig 1A⇓). Serum cholesterol levels did not differ significantly between the groups at the initiation of the study. Some increase in serum cholesterol was noted in both groups during the intermediate phase of the study, but the levels returned to the prior range by the end of the 9-month period. No significant differences in serum cholesterol were present between the two groups at any of the time points (Fig 1B⇓). Heart rate also was unaffected by trandolapril throughout the study (Fig 1C⇓).
Systolic blood pressures were comparable in the treatment groups before initiation of treatment. No significant changes were observed over the course of the study in either group, and no significant differences between groups were apparent at any time point (Fig 1D⇑).
Heart weight did not differ significantly in control (6.58±0.18 g) compared with trandolapril-treated (6.66±0.23 g) rabbits.
Serum ACE activity was significantly reduced by both the 10- and 250-μg/kg doses; the latter was used in our prior study.2 After 1 month of treatment, the serum ACE activity was decreased from 95.4±6.6 to 15.5 nmol · min−1 · mL−1 (P<.0001) in the low-dose group and to 2.8±0.2 nmol · min−1 · mL−1 (P<.0001) in the high-dose group (Fig 2A⇓). Aortic ACE also was significantly reduced in both treatment groups, decreasing from 0.590±0.026 nmol · min−1 · mg−1 protein in control rabbits to 0.115±0.014 (P<.001) and 0.100±0.010 nmol · min−1 · mg−1 (P<.001) in low- and high-dose groups, respectively (Fig 2B⇓).
Intimal Surface Involvement
No inhibition of surface involvement by atherosclerosis was apparent with trandolapril either in total aorta or in the individual regions of aorta. The average values for lesion area were actually somewhat greater in the trandolapril-treated than in the control group (Fig 3⇓), but the differences were not significant statistically.
Aortic cholesterol contents were not significantly different in the ascending aorta plus arch, descending thoracic aorta, or abdominal aortic segments (Fig 4⇓).
In both control and trandolapril-treated rabbits, representative lesions from descending thoracic aorta showed considerable thickening of the intima with numerous foam cells, increased connective tissue, and areas of necrosis and calcification. No qualitative differences in the nature of the lesions between control and treated rabbits were apparent. Medial thickness in the control WHHL rabbits (0.181±0.013 mm, mean±SEM) was not significantly different from that in trandolapril-treated animals (0.165±0.008 mm).
Since our original observations in the WHHL rabbit,1 2 several studies have indicated that ACE inhibitors can discourage atherosclerosis in cholesterol-fed animals. These have included rabbits given enalapril,3 monkeys treated with captopril,4 minipigs that received perindopril,5 and hamsters treated with fosinopril.6
In the present study, WHHL rabbits were treated with doses of trandolapril that were adequate to cause marked reductions in serum and aortic ACE activity but insufficient to lower blood pressure. The protocol was otherwise identical to the one we used previously in which much larger doses of trandolapril were administered, causing marked inhibition of atherosclerosis in association with blood pressure reduction. However, despite marked decreases in serum and arterial ACE activity, no antiatherosclerotic action of trandolapril could be demonstrated with the lower dose of the drug.
Hypertension is well recognized as a potent promoter of atherosclerosis in hypercholesterolemic animals13 14 and in humans.15 16 Epidemiological data have shown that human subjects with the lowest levels of blood pressure also have the lowest rate of clinical complications of atherosclerosis.16 17 Many of the arterial changes induced by hypertension mimic those caused by hypercholesterolemia, including impaired endothelium-dependent relaxation, increased arterial permeability, smooth muscle proliferation and accumulation, monocyte adherence to the endothelial surface, intimal macrophage accumulation, and connective tissue deposition.18 19 20 21 22 23 24 Despite these known effects of elevated blood pressure on the arterial wall, little data are available regarding the influence of mild reductions of blood pressure on the arterial responses to hypercholesterolemia. Prior studies from our laboratory suggested that age-related changes in arterial morphology may be reduced by blood pressure lowering,25 but the significance of these data with respect to atherogenesis is unknown.
In contrast to the present findings, recent studies in cholesterol-fed rabbits given enalapril3 and hamsters treated with captopril6 suggested that the protection against atherosclerosis by ACE inhibition in these models may be independent of blood pressure lowering. The reasons for the apparent differences in results between these and our own studies are unclear. In our experience, the blood pressures of WHHL rabbits have been consistently higher than those in control or cholesterol-fed New Zealand White rabbits, with systolic pressures in the WHHL animals typically ranging from 110 to 120 mm Hg,1 2 in contrast to the 80 to 90 mm Hg range in cholesterol-fed rabbits.26 In the enalapril studies,3 the mean blood pressure values for cholesterol-fed rabbits averaged 79 mm Hg. The higher blood pressures in WHHL rabbits might increase their predisposition to atherosclerosis, and blood pressure reduction by ACE inhibition might contribute to inhibition of plaque formation.
Aortic ACE activity was inhibited similarly (>80%) in the low- and high-dose trandolapril groups, even though only the high dose resulted in an antiatherosclerotic action. Serum ACE also was inhibited markedly in both groups, although the reductions were somewhat greater in the high-dose–treated animals. Although it is possible that our measurements of aortic ACE activity did not fully reflect the drug effects at the cellular level, the results suggest that the reduction of arterial and/or serum ACE activity alone does not account for the antiatherosclerotic action of ACE inhibitors in the WHHL rabbit.
Recent studies with the angiotensin II receptor antagonist SC-51316 failed to demonstrate any significant effect on atherogenesis in the cholesterol-fed rabbit,3 despite inhibition of pressor responses to infusion of both angiotensin I and angiotensin II. Other effects of ACE inhibition could play a role, particularly those related to the inhibition of kininase II and the potentiation of vascular bradykinin and prostacyclin. Bradykinin appears to participate in the action of ACE inhibitor–induced reduction of intimal hyperplasia after balloon injury of rat carotid artery.27 Enhancement of bradykinin can increase cGMP and the production of nitric oxide and prostacyclin by arterial endothelial cells.28 Such effects could lead to attenuation of platelet aggregation29 and of monocyte adherence and entry into the arterial intima.30 Stimulation of nitric oxide formation by l-arginine administration31 and treatment with a prostacyclin agonist32 has been reported to inhibit atherosclerosis in the cholesterol-fed rabbit. Thus, the effects of ACE inhibition on the development of atherosclerosis could be secondary to effects other than those related to reduced formation of angiotensin II.
This work was supported by grant HL-47124 (Hypertension SCOR) from the National Heart, Lung, and Blood Institute, National Institutes of Health, and by a grant from Knoll Pharmaceuticals.
- Received September 16, 1994.
- Revision received October 31, 1994.
- Accepted January 18, 1995.
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