Development and Progression of Atherosclerosis in Aorta From Heterozygous and Homozygous WHHL Rabbits
Effects of Simvastatin Treatment
Abstract This study was conducted to define progression of atherosclerosis in both homozygous and heterozygous Watanabe heritable hyperlipidemic (WHHL) rabbits and to investigate the ability of the HMG CoA reductase inhibitor simvastatin to attenuate progression of the disease. We examined contractile responses to phenylephrine and endothelium-dependent relaxation in response to carbachol in thoracic aorta at 3, 6, 9, and 12 months in control New Zealand White (NZW) rabbits, homozygous WHHL rabbits, and heterozygous WHHL rabbits. Homozygous and heterozygous rabbits were treated with simvastatin (10 mg/kg per day) from 3 to 6 months and from 9 to 12 months of age. Simvastatin significantly reduced serum cholesterol levels in young heterozygotes, with a nonsignificant trend toward a reduction in older heterozygotes. In homozygotes, no significant fall was observed. Contractile function declined progressively with age in all groups−most in homozygotes and least in NZW rabbits. Relaxation was unaffected by age in NZW rabbits; relaxation declined in the heterozygotes and declined to a greater extent in homozygotes. Simvastatin retarded the loss of function in the young heterozygotes. Similar trends were observed in young homozygotes and older heterozygotes, with no effect in older homozygotes. Histological studies revealed the progressive development of early atherosclerosis in heterozygotes, and more advanced atherosclerosis was observed in homozygotes. Simvastatin did not inhibit development of atheroma. A correlation was observed between vascular function and structure. However, functional changes preceded the development of atheroma. In addition, we have demonstrated that simvastatin can help to reduce the loss of vascular function associated with the progression of atherosclerosis in the heterozygous WHHL rabbit.
Presented in part to the British Pharmacological Society, September 1993, Rome, Italy.
- Received July 19, 1994.
- Accepted May 12, 1995.
Atherosclerosis is responsible for up to 75% of deaths in the Western world. Raised cholesterol levels are a major risk factor for the development of atherosclerosis. In addition to formation of fatty streaks and plaque, abnormalities in vascular functioning have been observed both in animals and in humans.1 2 3 Population studies have shown that lipid-lowering strategies are able to reduce the risk of developing atherosclerotic disease and myocardial infarction in particular. The HMG CoA reductase inhibitors are among the most promising classes of therapeutic agents available to date. Clinical trials with HMG CoA reductase inhibitors have shown beneficial effects on plasma LDL concentrations. Dose-dependent decreases in plasma levels of total and LDL cholesterol have been reported in patients with heterozygous familial hypercholesterolemia.4 5 6 In patients with homozygous familial hypercholesterolemia, the effectiveness of HMG CoA reductase inhibitors is dependent on whether the patient is receptor negative, in which case the inhibitors are ineffective,7 or receptor defective, in which moderate reductions in LDL cholesterol level are found.8 9 To date, in humans, the majority of these studies have only examined the effect of the HMG CoA reductase inhibitors on cholesterol levels, with little data available on the long-term efficacy of these compounds in reducing the development of atherosclerosis. Studies in animal models of atherosclerosis may be useful. Watanabe heritable hyperlipidemic (WHHL) rabbits develop atherosclerotic lesions very similar to those observed in humans.10 Like subjects with familial hypercholesterolemia, these animals have a defect of the LDL receptor.11 The resultant disease states in rabbits and humans are qualitatively similar with regard to deficiency of functional LDL receptors and the resultant altered lipid profiles. Numerous studies have been undertaken in WHHL rabbits,12 13 14 15 but because of poor breeding characteristics, WHHL rabbits are usually studied in small groups. Alternatively, wide age bands have been used to produce larger groups. In addition, most investigations in WHHL rabbits have focused on the homozygote, with very few studies in heterozygous animals despite the greater clinical prevalence of the heterozygous state in humans.
Several groups have conducted studies with HMG CoA reductase inhibitors in homozygous WHHL rabbits. Primarily, these studies have examined the effects of pravastatin on cholesterol levels.16 17 18 19 20 21 Three of these studies also examined the effect of pravastatin on the development of atheroma,17 18 21 whereas Fukuo and colleagues15 studied the effects of simvastatin on cholesterol levels and aortic atheroma. Only one study has examined vascular reactivity after lipid-lowering therapy.21 Furthermore, no published studies have examined the effect of lipid-lowering or antiatherogenic therapy in the heterozygous WHHL rabbits.
The aim of our study was therefore to define progression of atherosclerosis in the vasculature of both heterozygous and homozygous WHHL rabbits and to investigate the ability of the HMG CoA reductase inhibitor simvastatin to attenuate progression of the disease. Three broad aspects of the disease were examined: cholesterol levels, vascular function, and structural changes in both homozygous and heterozygous WHHL rabbits aged 3 to 12 months.
Plasma cholesterol of the WHHL rabbits was measured at weaning (8 to 10 weeks), and the animals were allocated to an appropriate experimental group. Animals with cholesterol levels of 1.5 to 5.0 mmol/L were designated as heterozygotes and those with cholesterol levels greater than 10 mmol/L as homozygotes.
Ten untreated groups of animals were studied: 3-, 6-, and 12-month-old heterozygous WHHL rabbits; 3-, 6-, 9-, and 12-month-old homozygous WHHL rabbits; 3-, 6-, and 12-month-old New Zealand White (NZW) rabbits. Male and female rabbits were included in the study. Groups were matched for sex (Table 1⇓).
In addition, the effects of treatment with simvastatin were examined in young rabbits treated from the age of 3 months until they were studied at 6 months of age and in an older group of rabbits treated from the age of 9 months until they were studied at the age of 12 months. These treatment groups were studied in both homozygous and heterozygous WHHL rabbit groups.
The simvastatin was administered orally at a dosage of 10 mg/kg per day. The drug was dissolved in ether and applied to cabbage leaves, the ether subsequently evaporated, and the drug was left as a coating on the cabbage leaf. The cabbage was fed to the rabbit and normally was eaten immediately.
Blood for plasma cholesterol was taken from a marginal ear vein just after weaning (8 to 10 weeks of age) and at 3, 6, 9, and 12 months of age in the WHHL rabbits and at study in NZW rabbits. Cholesterol was measured by the Boehringer Mannheim cholesterol C system, which is based on the CHOD-PAP method. The assays were carried out by the Biochemistry Department at Stobhill Hospital, Glasgow.
Rabbits were killed with an overdose of sodium pentobarbital (50 mg/kg) administered intravenously in the marginal ear vein. The thoracic aorta was immediately dissected out and immersed in ice-cold Krebs bicarbonate buffer of the following composition (mmol/L): NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, CaEDTA 0.05, glucose 11.1, pH 7.4. 17ß Estradiol (10−5 mol/L), cocaine hydrochloride (10−5 mol/L), and indomethacin (10−5 mol/L) were added to the Krebs buffer to block extraneuronal and neuronal adrenergic uptake and prostaglandin synthesis, respectively. The aorta was trimmed free of fat and adhering connective tissue. Transverse rings 2 mm wide were cut and suspended between two stainless steel hooks in a 10-mL organ bath filled with Krebs buffer at 37°C, continuously gassed with 95% O2 and 5% CO2. Two rings were obtained from each animal, and the responses were averaged.
Isometric tension was recorded using a force transducer (Grass model FT03) connected to a chart recorder (Grass polygraph model 7B). Each ring was set individually at the optimal point of its length-tension relation as determined by repeated exposure to phenylephrine (10−7 mol/L). Tissues were then allowed to equilibrate for 1 hour. Cumulative concentration curves to phenylephrine (10−8 to 10−5 mol/L) were obtained. The baths were washed out, and the tissues were allowed to reequilibrate for 45 minutes; they were then recontracted to between 50% and 70% of the maximum contraction to phenylephrine as determined from the full concentration response curve. Once a plateau contraction response was established, cumulative concentration response curves to carbachol (10−8 to 10−5 mol/L) were obtained. Before the baths were washed, each ring was exposed to 10−4 mol/L sodium nitroprusside (SNP). At the end of the assay, each ring was blotted dry and weighed.
Rings of thoracic aorta adjacent to those used for the assessment of functional studies were fixed in formalin and embedded in paraffin wax. They were subsequently sectioned and stained with elastic/Martius scarlet blue. The sections were examined and photographed with a light microscope (Leitz Orthoplan). We used image analysis to measure the internal circumference of the sectioned aortic ring and the internal elastic lamina as a guideline. The proportion of the intimal circumference occupied by atheroma was also measured and expressed as a percentage of total circumference. Image analysis was performed with use of the semiautomatic vids-v image analysis system. The remainder of the thoracic aorta was stained with Sudan IV, and the overall extent of atheroma was measured by planimetry.
Data are expressed as mean±SEM unless otherwise stated (n represents the number of rabbits studied). EC50 and EMAX were calculated individually for each concentration response curve using the equation where E is the contraction, EMAX is the maximum contraction, C is the concentration, EC is the EC50 (concentration of agonist required to induce half the maximum response), and m is Hill’s coefficient. Statistical analysis was carried out using repeated-measures ANOVA. Differences were considered to be significant at P<.05, and Bonferroni multiple comparisons were used where appropriate. Cholesterol levels and structural parameters were analyzed using t tests, allowing for multiple comparisons where necessary. Differences were considered to be significant at P<.05.
The materials used were obtained from the following suppliers: NZW rabbits, Interfauna, Wyton, Huntingdon, UK; WHHL rabbits, bred and maintained in-house at Stobhill Hospital, Glasgow. SNP and cocaine hydrochloride were from the pharmacy, Western Infirmary, Glasgow. Simvastatin was a generous gift from Merck Sharp and Dohme. All other chemicals and reagents were obtained from Sigma Chemical Co.
Cholesterol Levels in WHHL Rabbits
The mean plasma cholesterol levels at the age of 8 to 10 weeks of all the WHHL rabbit groups included in the study are shown in Table 1⇑. When compared with the cholesterol levels found in NZW rabbits, both heterozygous and homozygous groups were significantly elevated. Plasma cholesterol of the homozygous WHHL rabbits was massively elevated compared with the heterozygotes. The cholesterol level at the age of 8 to 10 weeks did not statistically differ between the four homozygous WHHL groups, nor was there any statistically significant difference in initial cholesterol levels between the three groups of heterozygous WHHL rabbits.
Plasma cholesterol levels at the time of study are also shown in Table 1⇑. It can be seen that plasma cholesterol fell between weaning and study in both the homozygous and heterozygous WHHL rabbits. Cholesterol levels in NZW rabbits did not differ significantly between groups (Table 1⇑).
Effects of Simvastatin Treatment on Cholesterol Levels
Treatment with simvastatin (10 mg/kg per day) had no significant effect on plasma cholesterol levels in homozygous WHHL rabbits (Table 1⇑). In the group treated from 3 to 6 months of age, there was a slight, nonsignificant fall in the treated group compared with the untreated group. In the older group treated with simvastatin from 9 to 12 months of age, there was little difference in the cholesterol levels at the time of study.
Treatment with simvastatin (10 mg/kg per day) led to a significant decrease in plasma cholesterol levels in heterozygous WHHL rabbits treated from 3 to 6 months of age when compared with the untreated 6-month-old group. In the older heterozygous WHHL rabbits treated with simvastatin, cholesterol levels were lower than in the age-matched control group; however, this just failed to reach statistical significance (Table 1⇑).
Functional Studies in the Thoracic Aorta of WHHL Rabbits
Changes in Phenylephrine-Induced Contraction With Increasing Age
In rings of thoracic aorta from homozygous WHHL there was a progressive decrease in the response to phenylephrine as the age of the animals increased. The maximum response was significantly reduced from the age of 3 months to the age of 6 months, with a further decrease in the level of contraction at 9 and 12 months of age (Table 2⇓). In heterozygous WHHL, there was also a decrease in aortic contractility associated with increasing age. As with the homozygotes, there was a statistically significant decrease in response to phenylephrine from the age of 3 to 6 months (Table 2⇓). There was no further change in the response to phenylephrine from the age of 6 to 12 months. Sensitivity to phenylephrine decreased slightly with age in both homozygous and heterozygous WHHL rabbits, illustrated by a small increase in EC50 values (Table 2⇓). To allow comparison with a normal control, rings of thoracic aorta from NZW rabbits were also studied. There was a small decrease in the levels of contraction in the thoracic aorta in response to phenylephrine as the age of the NZW rabbits increased; no change in sensitivity (EC50) was observed (Table 2⇓). This change in phenylephrine-induced contraction was taken to be representative of normal maturation.
Changes in Endothelium-Dependent Relaxation With Increasing Age
Aging was associated with a progressive loss of the ability of the thoracic aorta to respond to carbachol in homozygous WHHL (Table 2⇑). There was a statistically significant decrease in relaxation observed from 3 to 6 months of age, with a further decline in response at 9 and 12 months in the homozygous WHHL rabbits. At all ages, rings of thoracic aorta from homozygous WHHL rabbits were able to relax fully to 10−4 mol/L SNP. In heterozygous WHHL rabbit thoracic aorta there was also a decrease in the ability of the rings to relax in response to carbachol with increasing age (Table 2⇑). The decrease in vasorelaxation in response to carbachol at 6 months of age was statistically significant when compared with the 3-month group, but there was no further change in the response between 6 and 12 months. Due to the absence of any difference between the 6-month and 12-month heterozygous groups, a 9-month heterozygous group was not studied. As with the homozygous animals, all rings showed 100% relaxation when stimulated by 10−4 mol/L SNP. There was little or no change in carbachol-induced endothelium dependent relaxation in NZW rabbits of 3, 6 and 12 months of age (Table 2⇑). Age had no effect on sensitivity (EC50) in any of the groups studied.
Effects of Disease Status on Phenylephrine-Induced Contraction
To study the relative effects of the genetic LDL receptor deficiency in WHHL rabbits, the response to phenylephrine was compared in control NZW rabbits, heterozygous WHHL rabbits, and homozygous WHHL rabbits at three age points: 3, 6, and 12 months.
There were no statistically significant differences between the phenylephrine concentration response curves for the aortic rings from the control NZW rabbits and either the homozygous or the heterozygous WHHL rabbits at 3 months. However, responses from homozygous WHHL rabbits tended to be higher than those from either the control NZW rabbits or the heterozygous WHHL rabbits (Table 2⇑).
At 6 months there was no statistically significant difference in the responses of aortic rings to phenylephrine between the control NZW rabbits and the homozygous WHHL rabbits. The responses of the rings of aorta from heterozygous WHHL rabbits were significantly lower than those from both the control NZW rabbits and the homozygous WHHL rabbits (Table 2⇑).
At 12 months, the responses of rings of thoracic aorta from the heterozygous WHHL rabbits to phenylephrine were consistently less than those from NZW rabbits; however, this was not significant. The responses of aortic rings from homozygous WHHL to phenylephrine were significantly less than the responses from both the heterozygous WHHL rabbits and the NZW rabbits (Table 2⇑).
Effects of Disease Status on Endothelium-Dependent Relaxation
The responses from the 3-month-old homozygous WHHL rabbits were significantly less than the 3-month heterozygous WHHL and NZW rabbits. These differences became progressively greater at both 6 months and 12 months of age (Table 2⇑).
Effects of Simvastatin Treatment on Functional Responses in Thoracic Aorta From WHHL Rabbits
Phenylephrine-induced contraction. In homozygous WHHL rabbits treated with simvastatin from the age of 3 to 6 months, there was no significant improvement in maximum response to phenylephrine; however, the increase in EC50 was reduced (Table 2⇑). In homozygous WHHL rabbits treated with simvastatin from the age of 9 to 12 months, there was no significant effect of treatment. In heterozygous WHHL rabbits, simvastatin treatment significantly reduced the loss of phenylephrine-induced contraction that had been observed in thoracic aorta rings from the control group between the age of 3 to 6 months (Table 2⇑). In heterozygous WHHL rabbits treated from the age of 9 to 12 months there was a small but nonsignificant increase in contractile function.
Carbachol-induced relaxation. In homozygous WHHL rabbits treated with simvastatin from the age of 3 to 6 months, there was some improvement in function, that is, an increase in the level of relaxation in rings of thoracic aorta (Table 2⇑) such that the 6-month treated group was not significantly different from either the 3-month or the 6-month untreated group. In the homozygous rabbits treated with simvastatin from the age of 9 to 12 months, there was no effect on carbachol-induced relaxation of thoracic aorta rings when compared with the 12-month homozygous untreated group (Table 2⇑). In the heterozygous WHHL rabbits treated with simvastatin from the age of 3 to 6 months of age, simvastatin completely prevented the loss of carbachol-induced relaxation in the thoracic aorta (Table 2⇑). In the heterozygous WHHL rabbits treated with simvastatin from the age of 9 to 12 months, there was no significant difference when compared with the 12 month untreated group (Table 2⇑).
In the homozygous animals, there was a progressive increase with age in the amount of lipid visualized both macroscopically as Sudan IV red patches and also histologically in the aortic rings adjacent to those used in functional studies (Fig 1⇓). There was no identifiable atheroma at 3 months. At 6 months, sections from 4 of the 5 animals contained measurable amounts of atheromatous plaque. Atheromatous plaques were present in all sections from the 9-month-old homozygotes, and there was a statistically significant increase in the mean value of both percentage involved and Sudan staining when compared with the 6-month-old group. There was a slight further increase in the percentage involved by 12 months of age.
In the media, the changes were confined to the subintimal layer enclosed by the subjacent two or three elastic laminae. The earliest change, present in all animals at 6 months, was a focal increase in glycosaminoglycans. This was associated with some disturbance of the parallel orientation of a few subintimal smooth muscle cells. At 9 months, there was some further separation of the laminae that contained between them a few foam cells. In the most severe lesions at 12 months, the biggest plaques had eroded into the superficial media, leading to fragmentation of the inner two or three laminae only, most of the thickness of the media being normal. Unlike human atheroma, there was little or no inflammation. Typical examples demonstrating thickening of the intima, infiltration of lipids, and calcification are shown in Figs 2⇓ and 3⇓.
In the heterozygous animals, only one of the 6-month group contained an atheromatous plaque, the appearances being similar to the homozygotes at 6 months.
Correlation and Analysis of Studies in WHHL Rabbits
In the study of the development and progression of atherosclerosis in these WHHL rabbits, three general aspects have been examined: (1) cholesterol levels, (2) functional characteristics, and (3) structural characteristics.
To examine any possible relation among the three aspects of the disease state, variables were plotted against one another and where appropriate, linear correlation and regression were analyzed.
Relation Between Plasma Cholesterol Levels and Function of the Thoracic Aorta
There was no significant correlation between contractile function (maximum response to phenylephrine) and plasma cholesterol level at 3 or 6 months. However, at 12 months of age there was a significant correlation (Fig 4A⇓).
Plasma cholesterol levels and maximum carbachol-induced relaxation showed no significant correlation at 3 months. At 6 and 12 months of age, a significant correlation was observed between plasma cholesterol level and maximum carbachol-induced relaxation. At 6 months of age, the gradient of the line of regression was −1.85; by the age of 12 months, this gradient had become much steeper, with a value of −6.75. Thus, as the age of the animal increased, the effect of the plasma cholesterol on the ability of the tissue to relax increased (Fig 4B⇑).
Relation Between Cholesterol Levels and Structural Parameters
There was no detectable atherosclerosis at 3 months. At 6 months, there was a positive correlation between the amount of atherosclerosis and the plasma cholesterol level, with the line of regression having a gradient of 2.09. At 12 months of age, again there was a significant positive correlation, and the gradient (7.61) was steeper than at 6 months (Fig 4C⇑).
Relation Between Changes in Structure and Changes in Function
The amount of atherosclerosis (percentage of internal circumference involved) correlated significantly with the loss of contractile function. Thus, the greater the changes in structure, the less the vessel was able to contract. Similarly, the relation between the percentage involved and the relaxant properties of the vessel showed a significant correlation (Fig 5⇓).
Effect of Simvastatin on the Relations Among Cholesterol, Function, and Structure
After treatment with simvastatin, no significant relation was observed among cholesterol levels and contraction, relaxation, and percentage involved (Fig 4⇑). However, it can be seen that in the plots of function versus cholesterol, at 6 months of age (Fig 4⇑, A and B), the points representing treated animals tend to lie in the upper area of scatter, reflecting the improvements in function observed (Table 2⇑). However, this is not the case at 12 months of age, where little functional change was observed after simvastatin treatment, nor when structure is plotted versus cholesterol.
Significant correlations were observed when percentage involved after simvastatin treatment was plotted versus both the contractile response to phenylephrine and the relaxant response to carbachol. The line of regression was not significantly affected by treatment (Fig 5⇑).
The data presented represent a study designed to provide an overview of changes in function associated with the development and progression of atherosclerosis in both homozygous and heterozygous WHHL rabbits. To enable a comparison to a normocholesterolemic control, NZW rabbits also have been studied. There is limited information in the literature regarding heterozygous WHHL rabbits. However, the rationale for studying these animals is strong because in the human condition of familial hypercholesterolemia, the heterozygous state is a more common condition. Also, there is greater potential for clinical intervention because the disease does not produce symptomatic atherosclerosis until the fourth or fifth decade. Thus, the second aspect of this study investigates the potential beneficial effects of the HMG CoA reductase inhibitor simvastatin on the development and progression of atherosclerosis in both young and old, homozygous and heterozygous WHHL rabbits. The dosage of simvastatin used in this study is relatively high when compared with the dosage used clinically in humans. This is consistent with other studies in rabbits.17 18 21 It may simply reflect species differences between rabbits and humans with regard to the absorption and/or metabolism of simvastatin. However, one point to consider is that the elevations in plasma cholesterol concentrations observed in the homozygous WHHL rabbits (15 to 20 mmol/L) are relatively large even when compared with their human homozygous familial hypercholesterolemia counterparts (typical plasma cholesterol, 9.1 mmol/L).
Plasma cholesterol levels fell during maturation in homozygous WHHL rabbits, consistent with reports by others.14 15 There is little information regarding cholesterol levels in heterozygous WHHL rabbits, although one study by Esper et al22 reported no change in serum cholesterol levels in heterozygous WHHL rabbits during the first 18 months of life; by 2 years of age, they reported a small rise in cholesterol levels. This is consistent with our observations. As previously observed in homozygous WHHL rabbits, treatment with simvastatin had no significant effect on plasma cholesterol.15 In heterozygous WHHL rabbits, treatment with simvastatin was found to have a significant hypolipidemic effect in the young (3 to 6 months) but not the older group. There are no comparable data in the literature for our studies in the heterozygote WHHL rabbits. The fact that simvastatin was more effective at lowering the plasma cholesterol in the heterozygous WHHL rabbits supports the suggestion that HMG CoA reductase inhibitors act, at least in part, by increasing LDL receptor synthesis. Heterozygous WHHL rabbits have only one defective gene, thus any therapy promoting LDL receptor expression would be able to act effectively on the normal gene. In homozygous rabbits, where both genes are defective, increased transcription of a defective receptor will not produce any beneficial effect.
One of the primary aspects of this study was to investigate changes in function. In both the homozygous and heterozygous WHHL rabbits and NZW rabbits, phenylephrine-induced contraction of the thoracic aorta decreased with age. This trend was least apparent in the NZW rabbits and most dramatic in the homozygous rabbits. Other studies of various quality examining adrenergic contractile function exclusively in homozygous WHHL rabbit thoracic aorta have produced a range of results. Some groups, like ours, have observed a decreased adrenergic response,23 24 while others have observed no difference between WHHL and normocholesterolemic control rabbits.25 26
In our studies investigating relaxation to carbachol, the NZW rabbits showed little change up to 12 months of age. In contrast, in heterozygous WHHL rabbits, endothelium-dependent relaxation progressively declined with age, while an even more pronounced loss of response was observed in the homozygotes. Other groups investigating the relaxant properties of the thoracic aorta in homozygous WHHL rabbits have generally observed similar effects.12 14 23 24 26 As with the studies on contraction, there are virtually no data concerning the relaxation responses of the heterozygous animals.
The effects of simvastatin treatment on the functional responses of the aorta paralleled the changes in plasma cholesterol levels. In the homozygous WHHL rabbits, no significant protective effects were observed, but beneficial effects were observed in the heterozygous WHHL rabbits. In the control studies, no change in vasoactive function was observed from the age of 6 to 12 months in the heterozygous WHHL rabbits, and for this reason and problems associated with the breeding of sufficient animals, no 9-month control group was studied. As a consequence of this, valid statistical analysis cannot be carried out for the older (9- to 12-month) treatment group. However, the improved functional responses in the younger heterozygotes are clear. One previous study has examined vasoactive function after pravastatin treatment.21 In this study it was shown that 9 months of treatment with pravastatin (40 mg/kg per day) could prevent loss of coronary artery endothelium-dependent relaxation in homozygous WHHL rabbits. However, the treatment was ineffective against the loss of endothelium-dependent relaxation in the distal abdominal aorta.
The parallel changes occurring in the structure of the vasculature were related to the observed changes in the vasoactive function. Responses to agonists were related to the extent of atheroma: A negative relation between maximum response to phenylephrine and percentage involved was observed. However, the slope of the line of regression was shallow; thus, even when 100% of the intima was covered with atheroma, the vessel still had the ability to constrict. There was also a negative correlation between maximum carbachol-induced relaxation and percentage involved. Thus, the greater the development of atheroma, the greater the impairment of relaxation in response to carbachol. After administration of simvastatin, no significant change in this relation was observed. The line of regression is displaced upward, reflecting the fall in cholesterol levels; however, no change in gradient is observed. Thus, while the fall in cholesterol leads to an improvement in function, no effect on structure is observed. Previous studies with pravastatin were also unable to demonstrate prevention or regression of aortic atherosclerosis.17 18 21 However, two studies17 21 did show a decrease in coronary atheroma after long-term (>6 months) treatment with pravastatin, while the study by Khachadurian and coworkers18 was able to demonstrate a decrease in aortic cholesterol content.
The relation between plasma cholesterol levels and vascular function was also examined. From this data it can be seen that the changes observed in vascular function in WHHL rabbits are related to the elevated plasma cholesterol levels that result from the deficit of functional LDL receptors. After treatment with simvastatin, the observed improvement of function is reflected in the scatter of the data.
Comparable trends were observed when the relations between plasma cholesterol levels and the histological parameters were examined. Thus, the changes in vascular structure in WHHL rabbits were related to the elevated plasma cholesterol levels.
The mechanisms by which the vascular responses are impaired has not been directly examined in this study. The effects on the contractile responses are complex. A hyperreactivity to contractile agonists has been reported to occur under some conditions,23 25 27 and a slight increase in response to phenylephrine was seen in the 3-month homozygous animals in our study. However, as the animals increased in age, the hyperreactivity appeared to be overcome by the progression of the disease as the structural changes started to occur in the vessel. As the atheroma progressively intrudes into the media, causing disruption of the smooth muscle and elastic laminae, the ability of the vessel to constrict is impaired. This is seen most dramatically in the homozygous WHHL rabbits and to a lesser extent in the heterozygous WHHL rabbits, in which the atheroma is minimal. Migration of smooth muscle cells from the media to intima may also contribute to the observed loss of contractile function. It has been observed that smooth muscle cells, under certain conditions including atheroma, will alter their phenotype from the contractile status to the secretory status−thus, the overall contractile ability of the vessel segment as a whole may be impaired.
The effects of the disease on the relaxant properties of the vessel were also dramatic. Even in the initial stages of the disease, the ability of the vessel to relax in response to carbachol was impaired. This is illustrated in both the heterozygous and homozygous WHHL rabbits, in which carbachol-induced endothelium-dependent relaxation was impaired even at 3 months−a stage at which, structurally, the vessels are virtually normal. Changes in function before the manifestation of structural modification were also observed in aorta from homozygote WHHL rabbits by Wines and colleagues.24 This may indicate that some modification of the endothelium-derived relaxing factor–nitric oxide pathway is responsible for the impairment of endothelium-dependent relaxation. This modification is probably localized to the endothelium, as the experiments with SNP in this and other studies23 26 showed no change in responses to directly acting nitrovasodilators in the early stages of atherosclerosis.
The changes in vasoactive function that were observed in the WHHL rabbits can be reduced by cholesterol-lowering therapy. The fact that the functional changes in heterozygous WHHL rabbits can be prevented by a lipid-lowering therapy indicates that this modification in function is, at least in part, induced directly by the elevated cholesterol levels. In the homozygous animals, some retardation of the loss of function was observed. However, this was not paralleled by any decrease in the development of atheroma. Hence, the altered contraction and relaxation observed in homozygous relaxation are not simply due to the presence of atheroma, but as with the heterozygous animals, the elevation of plasma cholesterol is able to affect the vasoactive properties of the thoracic aorta.
As atherosclerosis develops in WHHL rabbits, a range of structural and functional changes takes place in the homozygous and heterozygous rabbits. The changes in function are related to the changes in structure. However, it should be noted that the vasoactive function of the aorta is modified before any macroscopic or microscopic changes were observed. This is most clearly demonstrated in the heterozygous WHHL rabbits which, until now, have been virtually excluded from the research in this field. In these animals, changes in both phenylephrine-induced contraction and carbachol-induced endothelium-dependent relaxation are seen in rings of thoracic aorta that appeared normal. The HMG CoA reductase inhibitor simvastatin is able to significantly retard the loss of vasoactive function associated with the development and progression of atherosclerotic disease in young heterozygous WHHL rabbits. Similar effects have been observed in young homozygous WHHL rabbits to a lesser extent, but not in the older animals. These effects were not paralleled by a decrease in the development of atheroma.
We would like to thank C.A. Howie for help and advice over statistical analysis, and the Department of Biochemistry, Stobhill General Hospital, for help in analyzing plasma cholesterol levels. We would also like to thank Merck Sharp and Dohme for the gift of simvastatin.
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