Research ArticleExperimental Studies
Open Access

Development of a Microminipig Model of Atherosclerosis for the Evaluation of a HMGCR Inhibitor

TOMONOBU YAMADA, HIROAKI KAWAGUCHI, AKI MATSUOKA, KOHEI AKIOKA, NAOKI MIURA, HIROYUKI IZUMI and AKIHIDE TANIMOTO
In Vivo January 2024, 38 (1) 98-106; DOI: https://doi.org/10.21873/invivo.13415

Abstract

Background/Aim: Atherosclerosis is known as a major risk factor for cardiovascular disease, and development of an animal model of atherosclerosis is required to investigate its clinical pathogenesis. We studied the optimal amount of cholesterol in the diet and the optimal experimental period for development of a Microminipig model of atherosclerosis for the evaluation of a hydroxymethylglutaryl-CoA reductase (HMGCR) inhibitor (atorvastatin). Materials and Methods: Eighteen male animals (3-4 months old) were divided into 3 groups. Group 1 consisted of control animals receiving a normal chow diet, Group 2 animals received a high fat (12% w/w) and low cholesterol (0.1% w/w) diet (HFLCD), and Group 3 animals received HFLCD+statin for 12 weeks. Animals received statin at 3 mg/kg body weight per day. HFLCD did not down-regulate the hepatic expression of HMGCR mRNA. Results: HFLCD increased body, omentum, and mesenteric adipose tissue weight, and induced hypercholesterolemia and atherosclerotic lesions in the abdominal aorta. HFLCD+statin inhibited hypercholesterolemia and atherosclerotic lesions, but not obesity. Conclusion: A microminipig atherosclerosis model induced by HFLCD can be used in the evaluation of HMGCR inhibitors for the treatment of hypercholesterolemia and atherosclerosis.

Key Words:

Atherosclerosis has been reported as a major risk factor in cardiovascular diseases, and arterial diseases are a leading cause of serious morbidity and mortality in the West (1). The westernization of diet in Japan in recent years may account for the increased incidence of coronary and cerebral artery diseases in the Japanese population (2). Both genetic and environmental factors have been linked to atherosclerosis, and therefore both should be investigated in a model that reflects the clinical pathogenesis.

The development of atherosclerosis models has been attempted in experimental animals, such as apolipoprotein E-deficient mice (3-5), Watanabe heritable hyperlipidemic (WHHL) rabbits (6-8), transgenic rabbits (9), and New Zealand White rabbits (10). Because swine have similar feeding habits, lipid metabolism, and circadian rhythm to humans, they are more suitable than rabbits or mice for analyzing the influence of environmental factors on atherosclerotic lesions, whereas, rabbits and mice differ from humans in lipid metabolism and some environmental factors (11-15). Domestic pigs have been used in previous research on physical treatments of the arteries due to their large blood vessels (16), but these animals are difficult to manage due to their size. In light of this difficulty, the Microminipig (MMPig) has emerged as an experimental animal model for non-clinical pharmacological and toxicological studies (17). The MMPig is the smallest of minipigs (e.g., Göttingen, Yucatan, and Clawn) available for experimental use (18). Recently, we established an MMPig atherosclerosis model induced by providing a high fat (12% w/w) and high cholesterol diet (0.2-5% w/w) (15, 19-21). In this model, dietary control alone was sufficient to induce atherosclerotic lesions similar to those seen in humans.

Statins [hydroxymethylglutaryl-CoA reductase (HMGCR) inhibitors] inhibit HMGCR via a rate-limiting enzyme in the mevalonate pathway and suppress blood cholesterol elevation in humans (22) and rabbits (23). They are, therefore, commonly used as therapeutic medication for hypercholesterolemia and atherosclerosis. Statins, such as atorvastatin, pravastatin, simvastatin, fluvastatin, pitavastatin, rosuvastatin generally have similar tolerability (24-26). To the best of our knowledge, statins have been evaluated in only a limited number of studies in swine (27-30).

In this study, we investigated the optimal amount of cholesterol in the diet and the optimal experimental period for development of an MMPig model of atherosclerosis for evaluation of an HMGCR inhibitor (atorvastatin).

Materials and Methods

Animals and diet. Eighteen male MMPigs aged 3-4 months (mean body weight=8.3±0.7 kg) were obtained from Fuji Micra Inc. (Shizuoka, Japan) and housed in a dedicated room at the Kagoshima University. The room was maintained in a laminar flow of filtered air at a temperature of 24±3°C, a relative humidity of 50±20%, and a 12-h light/dark cycle. Animals had free accessed to tap water and were provided a normal chow diet (NcD; Kodakara 73; Marubeni Nisshin Feed Inc., Tokyo, Japan) and a special diet on a daily basis. Animals were weighed once a week. All protocols were approved by the Ethics Committee of Animal Care and Experimentation at Kagoshima University (A09029) and the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd., Drug Safety Research Laboratories (IACUC 703-024), which is fully accredited by AAALAC International. Research was performed according to the Institutional Guidelines for Animal Experiments and in compliance with the Japanese Act on Welfare and Management of Animals (Act No. 105 and Notification No. 6).

Study design. Eighteen animals were divided into 3 groups (6 animals in each) and fed as follows for 12 weeks: Group 1 consisted of control animals fed NcD, Group 2 animals received a high fat (12% w/w, refined lard; Miyoshi Oil & Fat Co., Ltd., Tokyo, Japan) and low cholesterol (0.1% w/w, Wako Pure Chemical Industries, Ltd., Osaka, Japan) diet (HFLCD), and Group 3 animals received HFLCD with statin (HFLCD+Statin). The stain dose (atorvastatin, Pfizer Inc., New York City, NY, USA) was 3 mg/kg BW per day based on previous reports on humans and swine (27, 31). On the day following the final administration, all animals were deep-anesthetized (intramuscular injection of a mixture of 0.04 mg/kg medetomidine, 5 mg/kg ketamine hydrochloride, and 0.2 mg/kg midazolam) and then euthanized by exsanguination via the bilateral axillary arteries.

Hematology and biochemical analysis. Peripheral blood was drawn from the cranial vena cava at weeks 0, 2, 4, 6, 8, 10, and 12 to examine general hematology, blood chemistry, and lipoprotein profiling. The biochemical parameters measured consisted of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GTP), lactate dehydrogenase (LDH), total bilirubin, glucose, blood urea nitrogen, creatinine, and free-cholesterol (free-Cho). The levels of total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), and triglycerides (TG) were analyzed using an automated agarose gel electrophoresis apparatus (Epalyzer 2, Helena Laboratories, Saitama, Japan).

Measurement of blood oxidative stress. Serum derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) represent reactive oxygen metabolites and antioxidant capacity, respectively. Serum levels of these two markers were measured with a Free Carrio Duo Redox Analyzer system (Wismerll, Tokyo, Japan) equipped with a photometer and a thermostatically regulated mini-centrifuge, and both the d-ROM and BAP tests were conducted according to the manufacturer’s instructions. The BAP/d-ROMs ratio was also calculated.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR). The liver and small intestine were collected at necropsy and immediately stored in RNAlater, and total RNA was extracted from the liver and small intestine tissues using mirVana miRNA isolation kit (Invitrogen). The hepatic expression of LDLr, HMGCR, NPC1L1, SREBF1, SREBF2, SRB1, and LIPC mRNA, and the expression of APOBEC1 and NPC1L1 mRNA in the small intestine were quantified by qRT-PCR using TaqMan quantitative PCR analysis (Applied Biosystems, Oyster Bay, NY, USA), as previously reported (15). The primers and probes were from a predesigned gene expression assay or were designed based on the sequence information on the domestic swine (Applied Biosystems). The expression level of GAPDH mRNA was used as an internal control.

Pathology. At necropsy, the abdominal aorta, heart, liver, kidneys, spleen, and brain were removed from each animal. The omentum and mesenteric adipose tissue were weighed, and the weight relative to BW at necropsy was calculated. and the organs and abdominal aorta were cut open longitudinally and then fixed in 10% phosphate-buffered formalin. Then the aorta was stained with Oil-red O-stain. En face images of the aortae were captured with a digital camera. The Oil-red O-stained area relative to the whole surface area was calculated using Image J software. Additionally, the abdominal aortae were cut horizontally at 3-4 mm intervals and 18-24 pieces were embedded in paraffin, sectioned at 5 mm, and stained routinely with hematoxylin and eosin (HE). The area intima and vessel wall were then measured using Image J software (histomorphometry analysis), and the histopathological intima area/vessel area ratio (%) was calculated. The other fixed organs were also stained with HE using the same above method as described above and the prepared specimens were examined histopathologically.

Statistical analysis. All data were expressed as the mean±standard deviation (SD). Statistical analysis of the differences between groups (Group 1 vs. Groups 2 and 3, Group 2 vs. Group 3) was performed using one-way analysis of variance followed by the Tukey-Kramer multiple comparison test and Mann-Whitney U-test, and statistical analyses were performed using IBM SPSS Statistics 25 software (IBM, Tokyo, Japan). p<0.05 was considered statistically significant.

Results

Animal clinical aspects, body and adipose tissue weights. No abnormal animal conditions in clinical signs in any animal during the 12-week experimental period were noted. As shown in Figure 1, the BW gain in Groups 2 and 3 were significantly higher than in Group 1, while there were no significant differences between Groups 2 and 3. The absolute and relative weight of the omentum and mesenteric adipose tissue in Groups 2 and 3 showed significantly high values and/or a tendency toward high values compared to Group 1 while there were no significant differences between Group 2 and Group 3 (Table I). No statin-related inhibition of changes in these body and organ weight was noted.

Figure 1.
Figure 1.

Body weight gain (%) due to HFLCD feeding during a 12-week period. Body weight gain (%) at necropsy compared to Week 0. *p<0.05, **p<0.01 vs. Control. HFLCD: High fat and low cholesterol diet.

View this table:
Table I.

Abdominal fat tissue weight changes due to HFLCD feeding during 12-week period.

Hematology and blood biochemistry. Hematology analysis showed no anemia, leukopenia, or leukocytosis in any animal in Groups 2 or 3 during the experimental period when compared to Group 1 and the normal range for MMPigs in the reference data (18, 32).

Serum lipid metabolism parameters were analyzed (Figure 2). In comparison to Group 1, T-Cho, free-Cho, LDL-C, and HDL-C levels in Groups 2 and 3 increased rapidly, almost peaked in Weeks 2-4, and were maintained throughout the rest of the experimental period. Relevant levels in Group 3 decreased compared to those in Group 2. There were no significant differences in TG levels between the 3 groups, and the TG levels at all examined points in each animal were within the normal range for MMPigs in the reference data (18, 33). There were no abnormalities in other biochemical parameters including glucose, suggesting that there was no hyperglycemia, progressive hepatic, or renal injuries observed in any animal of Groups 2 or 3 when compared to Group 1 and the normal range for MMPigs in the reference data (18, 33).

Figure 2.
Figure 2.

Changes in serum lipid levels due to HFLCD feeding during a 12-week period. The serum total cholesterol (A), free-cholesterol (B), LDL-(C) and HDL- (D) cholesterol levels during 12-week period were demonstrated. *p<0.05, **p<0.01 vs. Control, #p<0.05, ##p<0.01 vs. HFLCD. LDL: Low-density lipoprotein, HDL: high-density lipoprotein, HFLCD: high fat and low cholesterol diet.

Measurement of blood oxidative stress. Compared to Group 1, d-ROMs were increased, and BAP and BAP/d-ROMs ratio was decreased in Groups 2 and 3 at the end of experiment (Figure 3).

Figure 3.
Figure 3.

Changes in the blood oxidative stress markers due to HFLCD feeding during a 12-week period. The serum d-ROMs (A) and BAP (B) levels, BAP/d-ROMs ratio (C) during 12-week period were demonstrated. *p<0.05, vs. Control (Group 1). d-ROMs: Derivatives of reactive oxygen metabolites, BAP: biological antioxidant potential, HFLCD: high fat and low cholesterol diet.

qRT-PCR at necropsy. Hepatic expression of LDLr, HMGCR, NPC1L1, SREBF1, SREBF2, SRB1, LIPC mRNA, and expression of APOBEC1 and NPC1L1 mRNA in the small intestine showed no significant differences between the 3 groups (Figure 4). However, the hepatic expression of HMGCR mRNA in Group 3 showed a tendency toward up-regulation compared to the expression in Groups 1 and 2 (Group 3 mean value: approximately 1.5-fold the Group 1 mean value, and approximately 2-fold the Group 2 mean value).

Figure 4.
Figure 4.

qRT-PCR analysis. Hepatic expression of LDLr (A), HMGCR (B), NPC1L1 (C), SREBF1 (D), SREBF2 (E), SRB1 (F), LIPC (G) mRNA and the expression of APOBEC1 (H) and NPC1L1 (I) mRNA in the small intestine at necropsy were quantified. G-1: Control, G-2: HFLCD, G-3: HFLCD+Statin. HFLCD: high fat and low cholesterol diet.

Hypercholesterolemia-induced atherosclerosis. En face analysis demonstrated that abdominal aortic atherosclerotic lesion areas were significantly increased in Group 2 compared to Groups 1 and 3 (Figure 5). In the histomorphometry analysis, the intima area/vessel area ratio in the abdominal aorta were significantly increased in Group 2 compared to Groups 1 and 3 (Figure 6). Aortic atheromatous plaque located at the intima was observed.

Figure 5.
Figure 5.

Pathology: En face analysis in abdominal aorta. (A) The atherosclerotic red-stained lesion areas (circle) in G-2 and G-3 detected by en face Oil-Red O stain. (B) The data of area ratio of Oil-red O stain (Oil-red O-stained area relative to whole surface area) was demonstrated. CA: Celiac artery; CMA: cranial mesenteric artery; RA: renal artery; OIA or IIA: outer or internal Iliac artery bifurcation; G-1: Control; G-2: HFLCD; G-3: HFLCD+Statin. **p<0.01 vs. Control, ##p<0.01 vs. HFLCD. HFLCD: High fat and low cholesterol diet.

Figure 6.
Figure 6.

Histomorphometory: Intima area/vessel area ratio. Atherosclerotic lesions in the abdominal aorta. (A) No abnormal changes were demonstrated (G-1). (B) Slight atheromatous plaque was demonstrated (G-2). (C) Very slight atheromatous plaque was demonstrated (G-3). (D) The data of intima area/vessel area (IA/VA) ratio (%) was demonstrated. VA: Vessel area; IA: intima area; G-1: Control; G-2: HFLCD; G-3: HFLCD+Statin. **p<0.01 vs. Control, #p<0.05 vs. HFLCD. HFLCD: High fat and low cholesterol diet.

Histopathological examination of other organs. No significant histopathological changes were observed in the heart, kidneys, spleen, brain, omentum, or mesenteric adipose tissue. Mild fatty degeneration was detected in the liver in Group 2 while not observed in Group 3 (data not shown). Despite the presence of atherosclerotic lesions, there was no thrombosis or spontaneous myocardial or cerebral infarction in any MMPig fed HFLCD.

Discussion

In MMPigs, it has been reported that hepatic expression of HMGCR mRNA is down-regulated by the feeding of a 12% w/w fat and 0.2-1.5% w/w cholesterol diet for 8 weeks, and that the hepatic expression of LDLR mRNA is down-regulated by the feeding of a 12% w/w fat and 0.5-1.5% w/w cholesterol diet for 8 weeks (15, 21). In the present study animals were given a diet configured to contain 12% w/w fat and 0.1% w/w cholesterol (HFLCD) for 12 weeks to evaluate the effects of a statin, and the model was judged successful, with no down-regulation of hepatic expression of HMGCR and LDLR mRNA. The dosage of statin was close to actual human intake, and although an increase in cholesterol was noted in blood chemistry, it was considered to be relatively mild. HFLCD in the present study induced significant hypercholesterolemia resulting in atherosclerosis of the abdominal aorta. Significant symptoms of metabolic syndrome (increased omentum and mesenteric adipose tissue weights) were also induced by HFLCD. It has been reported that statins do not reduce blood cholesterol in rats or mice, and rats and that therefore mice are not suitable as hyperlipidemia model animals (34). However, rabbits and MMPigs are judged to be suitable as model animals. In rabbits, statins up-regulate the hepatic expression of HMGCR and LDLR mRNA, and resultantly suppress blood cholesterol elevation (23). In the present study, the administration of low dose of statin suppressed serum T-Cho and LDL-C elevation, and this was considered to have been caused by up-regulation of HMGCR because the hepatic expression of HMGCR mRNA showed an increased tendency in MMPigs fed HFLCD+statin. The inhibition of hypercholesterolemia induction reduced atherosclerotic lesions in the abdominal aorta in MMPigs similar as in the previous rabbit report (10). On the other hand, up-regulation of hepatic expression of LDLR mRNA in MMPigs fed HFLCD+statin was thought to be related to species differences between pigs and rabbits. Because it has been reported that atorvastatin inhibits HMG-CoA reductase, but up-regulation of LDL receptors is not noted in miniature pigs (35). Moreover, LDLR−/− domestic pigs did not reveal inhibition of hypercholesterolemia by statin while LDLR+/− miniature pigs revealed (29, 30). Further study is necessary to clarify the relationship between hypercholesterolemia and LDLR genes in swine.

Statins raise serum HDL-C levels in humans; however, the potency of these actions varies with statins. In the present study, HFLCD+statin decreased serum HDL-C levels compared to the MMPig fed HFLCD alone. This phenomenon may have been caused by the potency of atorvastatin and/or species difference (24, 36-39). Further study is necessary to further understand these issues and confirm the findings.

Oxidative stress may be involved in various disorders and pathogeneses of human lifestyle diseases (40-42). Recent advances in detecting reactive oxygen species using serum d-ROMs and BAP have opened potential avenues in biomarker research. The d-ROMs production in circulation is associated with development of coronary artery disease (42), there is a negative correlation between blood BAP levels and carotid artery intima-media thickness (43), and it has been reported that blood BAP/d-ROMs ratios is low in obesity and metabolic syndrome (44). These blood oxidative stress markers can be considered useful for an MMPig atherosclerosis model because the changes in d-ROMs and BAP in the present study were similar to those in human atherosclerosis, although no effects of statin on blood oxidative stress were observed.

In conclusion, this MMPig atherosclerosis model induced by HFLCD will contribute to the evaluation of HMGCR inhibitors to hypercholesteromia and atherosclerosis.

Acknowledgements

This work was in part supported by a Health Labour Science Research Grant (No. 33361105) from the Ministry of Health, Labour and Welfare of Japan (to AT) and the Adaptable and Seamless Technology Transfer Program (A-Step No. AS2316907E) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to AT and HK). This work was also partly supported by JSPS KAKENHI Grant Numbers 16H05176 (to AT), 25450426, and 16K08023 (to HK).

Footnotes

  • Authors’ Contributions

    T.Y., H.K., A.M. and, K.A. collected sample material. T.Y., H.K., and A.T. planned the study; T.Y., A.M., and N.M. performed the experiments and analysed data; T.Y., H.K., A.M., H.I., and A.T. drafted the manuscript. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    There are no conflicts of interest in regard to the present study.

  • Received August 10, 2023.
  • Revision received September 22, 2023.
  • Accepted September 25, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Development of a Microminipig Model of Atherosclerosis for the Evaluation of a HMGCR Inhibitor
TOMONOBU YAMADA, HIROAKI KAWAGUCHI, AKI MATSUOKA, KOHEI AKIOKA, NAOKI MIURA, HIROYUKI IZUMI, AKIHIDE TANIMOTO
In Vivo Jan 2024, 38 (1) 98-106; DOI: 10.21873/invivo.13415
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