Protective Action of Simvastatin in the Development of Aortic and Hepatic Lesions in Hypercholesterolemic Rabbits

Introduction

As simvastatin is the most prescribed and distributed drug for high cholesterol by the National Health Service the authors carry out an experimental work to study special detailed effects of this drug [1]. With the use of hypocholesterolemic drugs, a reduction in clinical events is observed, explained by the relationship of lipid content and foamy cells of cleft plaques and the positive repercussions of this protective effect on plaques with “high risk” morphology. With such therapeutic intervention, such lesions are effectively stabilized and consequently, the incidence of clinical events is also decreased [2].

Several researchers have proven, through experimental investigations, that the reduction of blood lipids causes the regression of the atherosclerotic picture. After prolonged exposure to cholesterol-rich atherogenic diets, the animals now have plasma lipid levels higher than 600 mg/dl, with a large increase in coronary arterial collagen, elastin and cholesterol (most in esterified form). Later, when the animals switch to a vegetarian-type “regression diet”, plasma lipids fall rapidly to levels considered normal (140 mg/dl), but arterial lipids and connective tissue partially regress over 20 to 40 months. Histopathological evaluations, in turn, prove that the size of the atherosclerotic plaque is also reduced during this treatment regimen [3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16].

Clarkson, et al. [7], studying lipid-fed primates, investigated to which plasma cholesterol levels should be reduced in so that arterial lesions regress. By repeated manipulation of the diet of animals with atherosclerosis, with serial measurements of plasma cholesterol, cholesterol levels were close to 7.8 mmol/l (300 mg/dl) or 5.2 mmol/l (200 mg/dl) during the regression phase of the experiment. These findings showed that when plasma cholesterol was kept high, the progression of atherosclerosis was slower, being interrupted in the second year. However, when cholesterol was kept lower, there was a regression of arterial lesions, more markedly in the second year. Therefore, serum cholesterol levels close to 200 mg/dl do not lead to a growth of pre-existing lesions, on the contrary, and may lead to regression of the lesion.

Still, it is already well established that high plasma cholesterol levels pose a risk for the development of atherosclerotic disease. Therefore, lipid-reducing therapy is expected to reduce the risk of atherosclerosis and its consequences, especially coronary heart disease and its peripheral vascular complications. In the “Coronary Primary Prevention Study of Clinical Studies of Lipid Research”, it was found in hypercholesterolemic men that a 1% decrease in plasma cholesterol levels reduces the risk of ischemic heart disease by 2%. Extrapolating this correlation to the general population, treatment with cholesterol-lowering drugs should substantially reduce the risk of ischemic heart disease in patients with hypercholesterolemia.

The “vastatins” represented by lovastatin, simvastatin and pravastatin constitute a very effective class of medicines in the treatment of hypercholesterolemia. These substances have different degrees of inhibitory activity on the limiting enzyme of cholesterol synthesis (Hydroxy Methyl Coenzyme Reductase - HMG-CoA reductase) and also on specific liver receptors, increasing the catabolism of Low Density Lipoprotein Cholesterol (LDL-c).

Lovastatin, simvastatin and pravastatin are drugs absorbed by hepatocytes by intermediate carrier transport mechanisms, thus having access to HMG-CoA hepatic reductase [17]. In contrast, the uptake of these three drugs by peripheral cells occurs by passive diffusion. Pravastatin, because it has a hydrophilic property, significantly limits its penetration into the lipid membranes of peripheral cells, unlike lovastatin and simvastatin that can easily penetrate peripheral cells. Due to this characteristic, the access of pravastatin in the undesired sites is restricted, avoiding unwanted side effects. On the other hand, lovastatin and simvastatin, because they present penetration into extrahepatic tissues, may be more act at the level of the fatty plaque, allowing its regression [18, 19].

The introduction of lovastatin in the United States in 1987 [20], and simvastatin in Europe in 1989 [21, 22] marked a new era in the treatment of hypercholesterolemia, competitively inhibiting the enzyme HMG-CoA reductase that controls intracellular cholesterol synthesis [23].

The active form of simvastatin specifically inhibits HMG- CoA reductase, the enzyme that catalyzes the conversion of HMG to mevalonate in the cholesterol biosynthesis pathway. At therapeutic doses, however, the drug does not completely block conversion. Thus, mevalonate is available in the biologically necessary quantities. The inhibiting action of simvastatin, occurring at an early stage of the cholesterol biosynthesis pathway and being rapidly metabolized to acetyl-CoA, does not cause accumulation of potentially toxic steroids to the body [24].

In clinical studies, simvastatin has been shown to be effective in the marked reduction of total cholesterol levels (32%) in LDL (40%), moderate decrease in triglycerides (18%) and moderate increase in High Density Lipoprotein Cholesterol (HDL-c) (14%) [21, 25].

Simvastatin, significantly reducing blood cholesterol levels in humans, has a protective effect on the inhibition and regression of atherosclerosis as observed in studies in patients with family homozygous hypercholesterolemia [26]. In a study with 4,000 patients with ischemic heart disease and hypercholesterolemia treated with simvastatin, observed over a period of three years, there was a reduction in fatal or non-fatal myocardial infarction and sudden death (Scandinavian Study) [19].

Similar results were observed in rabbits with diet-induced hypercholesterolemia supplemented with cholesterol and treated with three concentrations of simvastatin, with a dose-dependent inhibitory effect by simvastatin on atherosclerosis. In this study, it was observed that animals treated with simvastatin prevented an increase in serum cholesterol level, inhibiting the increase of Very Low Density Lipoprotein (VLDL) and LDL-c. At the dose of 10 mg/kg, the HDL-c level was slightly reduced compared to the control, but these values were significantly higher than the control group without an atherogenic diet. In this dose, inhibition of total cholesterol increase was found by 96%, which may be the cause of HDL-c elevation inhibition. As these animals presented a hyperlipidemia due to the diet, therefore an exogenous source, with a study of absorption of radiative cholesterol, the authors observed that simvastatin inhibited the absorption of cholesterol from the gastrointestinal wall, thus preventing exogenously induced hypercholesterolemia. It was also observed that the concentration of total cholesterol in the aortic wall was lower in the group treated with simvastatin at the concentration of 10 mg/kg and also prevented the development of atherosclerosis in the aorta and coronary arteries. Thus, the hypolipidemic effect of simvastatin is the main mechanism by which the development of atherosclerosis is inhibited [27].

Studying the action of three HMG-CoA reductase inhibitors (Lovastatin, Simvastatin, Pravastatin) in hypercholesterolemia and atherosclerosis in rabbits developed hyperlipidemic through diet, Thiery, et al. [28, 29] found lower concentration of beta-Very Low Density Lipoprotein (beta-VLDL) in treated animals. However, the study of the aortas showed fewer lesions and lower concentration of tissue cholesterol in animals treated with simvastatin and lovastatin.

In rabbits with hereditary hyperlipidemia (WHHL), Fukuo et al. [30], studied the correlation between lipid concentration and atherosclerosis in animals treated with simvastatin. In this study, rabbits at 3 and 10 months of age were used receiving simvastatin daily for 24 weeks, with a significant reduction in serum levels of total cholesterol, triglycerides and beta lipoproteins, with greater reduction in the group of younger animals. Also in this group, the extent of atheromatous lesions of the thoracic and abdominal aortas was smaller in the treated animals than in the control group. Therefore, the use of cholesterol-lowering drugs in hypercholesterolemic patients is indicated as a preventive measure against the development of atherosclerosis, as has been demonstrated in clinical and experimental studies.

Discussion

There are numerous contributions of animal models in atherosclerosis studies. A large number of studies were conducted to better understand the pathogenesis and consequently the therapy of the disease for later application in humans.

The choice of the animal model to be used depends on the variable to be studied, with attention to studies that correlate the type of lesion to be induced and the susceptibility of the animal species.

In our study, we used rabbits fed cholesterol-rich diet as a model for the study of the development of atherosclerosis. Although rabbit is the animal most frequently used in atherosclerosis research, many researchers have criticized the use of this animal, because the lesions result from extremely lipidemic conditions, with topography and the type of lesions different from those found in man. However, the use of different types of diet has induced different types of lesions, some of them closer to human lesions [33].

In the induction of hyperlipidemia, through the hypercholesterolemic diet, different concentrations of cholesterol have been used. In our study, we used the methodology described by Badimon, et al. [15], but using the cholesterol concentration of 1.5%, according to the model developed by Thomaz [34], obtaining significant atherosclerotic lesions after nine weeks of diet. Through this model, we analyzed the effect of simvastatin on the development of atherosclerotic lesion. Simvastatin is a hypercholesterolemic agent, being effective in reducing total cholesterol and LDL levels, inhibiting the action of the enzyme HMG-CoA reductase. With the inhibition of this enzyme, there is a depletion of liver cholesterol, causing a compensatory increase in the number of hepatic LDL-c receptors, concomitant removal of LDL for circulation and decrease of plasma cholesterol.

When in our study we dosed serum cholesterol, we verified similar initial levels of total cholesterol, with no significant difference between the groups (52, 42 and 45 mg/dl, respectively for groups A, B and C). However, after the fifth week, we observed a significant increase in the animals of group A, fed cholesterol, as well as group B, treated with simvastatin (424 and 661 mg/dl). After nine weeks, while in group A the cholesterol level increased significantly (1483 mg/dl), in group B it remained close to that of the fifth week (661 mg/dl) (Table 1). These results are close to those obtained by Badimon, et al. [15], who observed total serum cholesterol around 1559 mg/dl, as well as those obtained by Cotrim [35] of 1342 mg/dl although using a diet with cholesterol at 0.5% and for a period of 90 days. Jorge, et al. [36] reported levels of 1907 mg/dl after eight weeks of hypercholesterolemic diet.

Our study was in agreement with Thomaz [34], who used a diet with 1.5% cholesterol and found cholesterol in the fifth week, 1084 md/dl and in the ninth week 1469 mg/dl. Our results are also compatible with Uint [37] who administered a 1.0% hypercholesterolemic diet for 15 weeks to a group of rabbits, in a comparative study of varied diets, obtaining hypercholesterolemia and subsequent induction of thoracic aortic lesions.

(T0 X T5 X T9) X2 critical = 5,99

The use of simvastatin in hypercholesterolemic rabbits, whether induced by diet or genetically hyperlipidemic, has been studied by other authors, whose results are in agreement with some of the parameters analyzed by us. Thus, Fukuo, et al. [30] found a significant reduction in total serum cholesterol, LDL cholesterol and triglycerides, after administering simvastatin at a dose of 10 mg for 24 weeks to genetically hyperlipidemic Watanabe rabbits. A decrease in the installation of lesions in the thoracic and abdominal aorta was observed only in young animals.

Jorge, et al. [36] reported preservation of endothelial function and improvement of the dose-response curve to acetylcholine, both with simvastatin and pravastatin in hypercholesterolemic rabbits. In our study, we used the simvastatin dosage of 10 mg/day/rabbit, corresponding to approximately 3 mg/kg of weight and verified with this dosage a lower serum cholesterol level in the group that received the drug than in the group that received only the hypercholesterolemic diet.

Kobayashi, et al. [27] administering different doses of simvastatin (2.5, 5 and 10 mg/kg/day) for three months to rabbits fed 1% cholesterol, found that the cholesterol level was reduced from 2263 mg/dl to 981 mg/dl, 445 mg/dl, 94 mg/dl, respectively, and aortic involvement with atheromatous lesions was 18.7%, 8.4% and 0%, compared with 43.5% of the untreated group.

In addition to the cholesterol level, we also dose the serum triglyceride level, in which we see a slight increase in the cholesterol-fed groups. After nine weeks, we found a statistically significant increase in the two groups with hyperlipidemic diet (group A = 153 mg/dl, group B = 113 mg/dl and group C = 49 mg/dl) (Table 2). However, this increase may be due to the methodology used in the assays (automated methodological analysis), which is influenced by serum turbidity.

(T0 X T5 X T9) X2 critical = 5,99

Regarding the enzymatic assays of transaminases and GGT, we observed in group B alterations to a lesser degree than the group that received only cholesterol (group A). This variation may have been due to hepatic intracellular metabolism being overloaded by lipid accumulation. The transaminases assays were less altered in the group treated with simvastatin, due to perhaps the lower increase in the level of circulating cholesterol in this group and consequently, lower liver overload. Through the histopathological study, we also observed slightly less marked liver alterations than in the group treated with the drug.

For indirect follow-up of the clinical and nutritional evolution of the animals, we performed hematological measurements: blood count, hematocrit, white blood cell count and platelets. Although variability was observed in hematometric measurements, they do not compromise the subsidiary laboratory information of the follow-up of the clinical evolution of the animals. These assays showed large individual variations, serving, however, as laboratory support to monitor the clinical evolution of the animals. Hemoglobin values were 9.5 g/dl, 11.0 g/dl, 14.0 g/dl, and also groups A and B presented lower hematocrit values at the end than at the beginning of the experiment, but did not reach anemia levels.

Silver, et al. [38] describe the possibility of anemia in rabbits submitted to a hypercholesterolemic diet. The authors report hemolytic anemia and acanthocytosis and hepatic hemosiderosis in rabbits with a high-cholesterol diet for more than a year, mainly in dogs and pigs. We also observed an increase in the number of white blood cells in the three groups and similarity in the number of platelets between the groups.

The most relevant findings relate to the evaluations of aortic lesions, observing lesions in greater number and intensity in the region of the aortic arch of group A animals, being less pronounced in the more distal parts of the artery. In group B, the lesions were moderate with distinctly lower, smoother and sparse plaques.

We added to the microscopic morphometric analyses, where we could observe intense lipid content in macrophages, discriminating the degree of associated fibrosis, according to groups A: B, and C. The regression of thickening of the intima in the treated group, of 146 μm and 83 μm in the thoracic and abdominal aortas, respectively, in relation to 313 μm and 148 μm in the group without medication, confirm the findings of several researchers regarding the protection of the vessel and endothelium in particular. We believe it is appropriate to introduce, at this point, a resumption of the issue from the point of view of the literature in general.

A place of synthesis, the endothelium can be considered the largest paracrino organ of the organism, which responds to various stimuli producing and locally secreting a large number of metabolically active compounds that act in neighboring cells [39]. Among the multiple biological functions of the endothelium, the following stand out [40]:

1. Synthesis of connective tissue components such as collagen, elastin, proteoglycans (heparin, heparin- sulfate) and glycoproteins (linin, fibronectin, thrombospondin, factor VIII - von Willebrand complex);
2. Production of anticoagulant, fibrinolytic factors (eg, tissue plasminogen activator - tPA) and procoagulants;
3. Secretion of mitogenic factors;
4. Synthesis of relaxing substances of smooth muscle cells (vasodilators) as a relaxation factor of the endothelium (EDRF), hyperpolarizing factor derived from the endothelium (EDHF) and prostacyclin;
5. Synthesis of vasoconstrictor substances, in response to appropriate stimuli: endothelins, endoperoxides,
6. Receptor-modulator of vasoactive substances and involved in platelet aggregation and coagulation: acetylcholine, serotonin, thrombin, adenosine nucleotides, arachidonic acid, bradykinin, vasopressin;
7. Metabolism and inactivation of catecholamines, serotonin and other platelet products.
8. Hypercholesterolemia influences vascular reactivity and endothelium, i.e., in the presence of hypercholesterolemia, the vessel responds to certain stimuli with vasoconstriction instead of vasodilation.

When atherosclerosis regression is induced in experimental animals, the vessel reacts normally again, that is, it produces vasodilation under the action of acetylcholine and platelets. In this process, the integrity of the endothelium is always necessary.

The regression of atherosclerosis, therefore, becomes a process of great importance, especially for two basic aspects: 1. Plaque regression, no matter how small, significantly alters the resistance of the stenosis and favors the restoration of blood flow; 2. The reduction of hypercholesterolemia restores the normal vasodilator capacity of the vessel, provided that the endothelium remains intact.

Atheromatous plaques may assume several morphological characteristics compromising the artery, modifying vasomotor tone or obstructing vessel light. It can occupy an eccentric position located only in an arc of the circumference of the vessel or concentric, involving the entire circumference. As the eccentric plates do not affect the entire circumference of the vessel, with a residual arch with the medial structure intact, it maintains the ability to vary vascular tone. Plaques that are not yet visible angiographically are mainly eccentric, and the frequency of this type of lesion is lower the higher the degree of risk of stenosis. Many patients with unstable angina have at least one stenosis with more than 50% of the vessel diameter. In addition to the location of the plaque in the vessel wall, the severity of stenosis may be dependent on plaque constitution with regard to the relative proportion between lipid content and collagen. In general, lesions of high degree of stenosis (> 70% of the affected diameter) are predominantly collagenous, with lower lipid content and histologically complex, with proliferation of smooth muscle cells [41].

Atherosclerotic lesion is therefore characterized by an accumulation of cells overloaded with esterified cholesterol (foamy cells), underlying the arterial endothelium. Most of these cells are derived from circulating monocytes that turn into resident macrophages, although some are derived from arterial smooth muscle cells that migrate from the media to the intima and undergo transformations.

The morphometry of fatty plaques in histological sections revealed to be the source of the most original data of the present investigation, because the affected area of the aorta and the topography of its lesions did not discriminate the two hypercholesterolemic groups. If we observed, however, for the heights of the lesions and consequently their volumes, they presented significantly lower values in the group that was treated with simvastatin.

By the light of even more recent studies [36], the regression observed in the plate, whether at the area or height level, occurs basically due to the decrease in circulating LDL-c levels. In our case, in which we used simvastatin, this happened in the measurement of height and, consequently, the volume of lesions. We believe we can infer, therefore, that the lipid composition in the plates also decreased, because we worked with young plates, of recent formation, corroborated by the reports of histological analyses.

The influence of simvastatin in the process resulting from its intracellular action of inhibition of cholesterol synthesis was to reduce cholesterol concentration, stimulate the appearance of receptors on the surfaces of cell membranes, or to restore the catabolism function of LDL lipoproteins, with a reduction of cholesterolemic levels of

44.6%. This mechanism was triggered by simvastatin in a condition in which hypercholesterolemia caused by diet had already practically saturated the response of LDL receptors, as explained by Brown & Goldstein [42] in a “mimicry” of the genetic condition of family hypercholesterolemia. Extrapolation to the clinical reasoning of this work done in fibroblast culture, plus that of the animal model used in this investigation obviously deserves caution, however, it is justified by the confrontation with absolutely concordant data in man, obtained in multicenter studies and arteriographic regression studies, such as those reported and compiled by Brown, et al. [43].

Finally, we believe it is worth emphasizing that the improvements evaluated by laboratory analyses and histological measurements, through the use of simvastatin, can revert into other benefits, such as plaque stabilization and endothelial tonic function better restored.

Conclusion

• We found that the group of rabbits treated with hypercholesterolemic diet and simvastatin presented, after nine weeks, a significantly lower cholesterol level than the group not treated with the drug.
• The surface of the aorta compromised with fatty lesions was similar in both hypercholesterolemic groups.
• There was a protective effect by simvastatin, evaluated by the thickness of the atheroma, with a fatty plaque with lower height in the intima layer of the aorta.
• In the liver histological analyses of animals with a hypercholesterolemic diet, we observed intense morphological changes with hydropic degeneration and steatosis, although to a slightly lower degree in the group treated with the drug.