HDL quality features revealed by proteome‒lipidome connectivity are associated with atherosclerotic disease.

Lipoprotein, especially high-density lipoprotein (HDL), particles are composed of multiple heterogeneous subgroups containing various proteins and lipids. The molecular distribution among these subgroups is closely related to cardiovascular disease (CVD). Here, we established high-resolution proteomics and lipidomics (HiPL) methods to depict the molecular profiles across lipoprotein (Lipo-HiPL) and HDL (HDL-HiPL) subgroups by optimizing the resolution of anion-exchange chromatography and comprehensive quantification of proteins and lipids on the omics level. Furthermore, based on the Pearson correlation coefficient analysis of molecular profiles across high-resolution subgroups, we achieved the relationship of proteome‒lipidome connectivity (PLC) for lipoprotein and HDL particles. By application of these methods to high-fat, high-cholesterol diet-fed rabbits and acute coronary syndrome (ACS) patients, we uncovered the delicate dynamics of the molecular profile and reconstruction of lipoprotein and HDL particles. Of note, the PLC features revealed by the HDL-HiPL method discriminated ACS from healthy individuals better than direct proteome and lipidome quantification or PLC features revealed by the Lipo-HiPL method, suggesting their potential in ACS diagnosis. Together, we established HiPL methods to trace the dynamics of the molecular profile and PLC of lipoprotein and even HDL during the development of CVD.

Development of the Nude Rabbit Model.

Loss-of-function mutations in the forkhead box N1 (FOXN1) gene lead to nude severe combined immunodeficiency, a rare inherited syndrome characterized by athymia, severe T cell immunodeficiency, congenital alopecia, and nail dystrophy. We recently produced FOXN1 mutant nude rabbits (NuRabbits) by using CRISPR-Cas9. Here we report the establishment and maintenance of the NuRabbit colony. NuRabbits, like nude mice, are hairless, lack thymic development, and are immunodeficient. To demonstrate the functional applications of NuRabbits in biomedical research, we show that they can successfully serve as the recipient animals in xenotransplantation experiments using human induced pluripotent stem cells or tissue-engineered blood vessels. Our work presents the NuRabbit as a new member of the immunodeficient animal model family. The relatively large size and long lifespan of NuRabbits offer unique applications in regenerative medicine, cancer research, and the study of a variety of other human conditions, including immunodeficiency.

Human apolipoprotein A-II reduces atherosclerosis in knock-in rabbits.

BACKGROUND AND AIMS: Apolipoprotein A-II (apoAII) is the second major apolipoprotein of the high-density lipoprotein (HDL) particle, after apoAI. Unlike apoAI, the biological and physiological functions of apoAII are unclear. We aimed to gain insight into the specific roles of apoAII in lipoprotein metabolism and atherosclerosis using a novel rabbit model. METHODS: Wild-type (WT) rabbits are naturally deficient in apoAII, thus their HDL contains only apoAI. Using TALEN technology, we replaced the endogenous apoAI in rabbits through knock-in (KI) of human apoAII. The newly generated apoAII KI rabbits were used to study the specific function of apoAII, independent of apoAI. RESULTS: ApoAII KI rabbits expressed exclusively apoAII without apoAI, as confirmed by RT-PCR and Western blotting. On a standard diet, the KI rabbits exhibited lower plasma triglycerides (TG, 52%, p < 0.01) due to accelerated clearance of TG-rich particles and higher lipoprotein lipase activity than the WT littermates. ApoAII KI rabbits also had higher plasma HDL-C (28%, p < 0.05) and their HDL was rich in apoE, apoAIV, and apoAV. When fed a cholesterol-rich diet for 16 weeks, apoAII KI rabbits were resistant to diet-induced hypertriglyceridemia and developed significantly less aortic atherosclerosis compared to WT rabbits. HDL isolated from rabbits with apoAII KI had similar cholesterol efflux capacity and anti-inflammatory effects as HDL isolated from the WT rabbits. CONCLUSIONS: ApoAII KI rabbits developed less atherosclerosis than WT rabbits, possibly through increased plasma HDL-C, reduced TG and atherogenic lipoproteins. These results suggest that apoAII may serve as a potential target for the treatment of atherosclerosis.

Glycine-based treatment ameliorates NAFLD by modulating fatty acid oxidation, glutathione synthesis, and the gut microbiome.

Nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH) has reached epidemic proportions with no pharmacological therapy approved. Lower circulating glycine is consistently reported in patients with NAFLD, but the causes for reduced glycine, its role as a causative factor, and its therapeutic potential remain unclear. We performed transcriptomics in livers from humans and mice with NAFLD and found suppression of glycine biosynthetic genes, primarily alanine-glyoxylate aminotransferase 1 (AGXT1). Genetic (Agxt1 -/- mice) and dietary approaches to limit glycine availability resulted in exacerbated diet-induced hyperlipidemia and steatohepatitis, with suppressed mitochondrial/peroxisomal fatty acid ß-oxidation (FAO) and enhanced inflammation as the underlying pathways. We explored glycine-based compounds with dual lipid/glucose-lowering properties as potential therapies for NAFLD and identified a tripeptide (Gly-Gly-L-Leu, DT-109) that improved body composition and lowered circulating glucose, lipids, transaminases, proinflammatory cytokines, and steatohepatitis in mice with established NASH induced by a high-fat, cholesterol, and fructose diet. We applied metagenomics, transcriptomics, and metabolomics to explore the underlying mechanisms. The bacterial genus Clostridium sensu stricto was markedly increased in mice with NASH and decreased after DT-109 treatment. DT-109 induced hepatic FAO pathways, lowered lipotoxicity, and stimulated de novo glutathione synthesis. In turn, inflammatory infiltration and hepatic fibrosis were attenuated via suppression of NF-κB target genes and TGFß/SMAD signaling. Unlike its effects on the gut microbiome, DT-109 stimulated FAO and glutathione synthesis independent of NASH. In conclusion, impaired glycine metabolism may play a causative role in NAFLD. Glycine-based treatment attenuates experimental NAFLD by stimulating hepatic FAO and glutathione synthesis, thus warranting clinical evaluation.

Macrophage-derived MMP-9 enhances the progression of atherosclerotic lesions and vascular calcification in transgenic rabbits.

Matrix metalloproteinase-9 (MMP-9), or gelatinase B, has been hypothesized to be involved in the progression of atherosclerosis. In the arterial wall, accumulated macrophages secrete considerable amounts of MMP-9 but its pathophysiological functions in atherosclerosis have not been fully elucidated. To examine the hypothesis that macrophage-derived MMP-9 may affect atherosclerosis, we created MMP-9 transgenic (Tg) rabbits to overexpress the rabbit MMP-9 gene under the control of the scavenger receptor A enhancer/promoter and examined their susceptibility to cholesterol diet-induced atherosclerosis. Tg rabbits along with non-Tg rabbits were fed a cholesterol diet for 16 and 28 weeks, and their aortic and coronary atherosclerosis was compared. Gross aortic lesion areas were significantly increased in female Tg rabbits at 28 weeks; however, pathological examination revealed that all the lesions of Tg rabbits fed a cholesterol diet for either 16 or 28 weeks were characterized by increased monocyte/macrophage accumulation and prominent lipid core formation compared with those of non-Tg rabbits. Macrophages isolated from Tg rabbits exhibited higher infiltrative activity towards a chemoattractant, MCP-1 in vitro and augmented capability of hydrolysing extracellular matrix in granulomatous tissue. Surprisingly, the lesions of Tg rabbits showed more advanced lesions with remarkable calcification in both aortas and coronary arteries. In conclusion, macrophage-derived MMP-9 facilitates the infiltration of monocyte/macrophages into the lesions thereby enhancing the progression of atherosclerosis. Increased accumulation of lesional macrophages may promote vascular calcification.

Lipid Profiling of Serum and Lipoprotein Fractions in Response to Pitavastatin Using an Animal Model of Familial Hypercholesterolemia.

Statins are widely used for the treatment of atherosclerotic cardiovascular diseases. They inhibit cholesterol biosynthesis in the liver and cause pleiotropic effects, including anti-inflammatory and antioxidant effects. To develop novel therapeutic drugs, the effect of blood-borne lipid molecules on the pleiotropic effects of statins must be elucidated. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for hypercholesterolemia, are suitable for the determination of lipid molecules in the blood in response to statins because their lipoprotein metabolism is similar to that of humans. Herein, lipid molecules were investigated by lipidome analysis in response to pitavastatin using WHHLMI rabbits. Various lipid molecules in the blood were measured using a supercritical fluid chromatography triple quadrupole mass spectrometry. Cholesterol and cholesterol ester blood concentrations decreased by reducing the secretion of very low density lipoproteins from the liver. Independent of the inhibition effects of cholesterol biosynthesis, the concentrations of some lipids with anti-inflammation and antioxidant effects (phospholipid molecules with n-6 fatty acid side chains, lysophosphatidylcholines, phosphatidylethanolamine plasmalogens, and ceramide molecules) were significantly altered. These findings may lead to further investigation of the mechanism of statin action.

Identification of novel serum markers for the progression of coronary atherosclerosis in WHHLMI rabbits, an animal model of familial hypercholesterolemia.

BACKGROUND AND AIMS: The development of serum markers specific for coronary lesions is important to prevent coronary events. However, analyses of serum markers in humans are affected by environmental factors and non-target diseases. Using an appropriate model animal can reduce these effects. To identify specific markers for coronary atherosclerosis, we comprehensively analyzed the serum of WHHLMI rabbits, which spontaneously develop coronary atherosclerosis. METHODS: Female WHHLMI rabbits were fed standard chow. Serum and plasma were collected under fasting at intervals of 4 months from 4 months old, and a total of 313 lipid molecules, 59 metabolites, lipoprotein lipid levels, and various plasma biochemical parameters were analyzed. The severity of coronary lesions was evaluated with cross-sectional narrowing (CSN) corrected with a frequency of 75%-89% CSN and CSN> 90%. RESULTS: There was a large variation in the severity of coronary lesions in WHHLMI rabbits despite almost no differences in plasma biochemical parameters and aortic lesion area between rabbits with severe and mild coronary lesions. The metabolites and lipid molecules selected as serum markers for coronary atherosclerosis were lysophosphatidylcholine (LPC) 22:4 and diacylglycerol 18:0-18:0 at 4 months old, LPC 20:4 (sn-2), ceramide d18:1-18:2, citric acid plus isocitric acid, and pyroglutamic acid at 8 months old, and phosphatidylethanolamine plasminogen 16:1p-22:2 at 16 months old. CONCLUSIONS: These serum markers were coronary lesion-specific markers independent of cholesterol levels and aortic lesions and may be useful to detect patients who develop cardiovascular disease.

Widely-targeted quantitative lipidomics method by supercritical fluid chromatography triple quadrupole mass spectrometry.

Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylamine-bonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery.-Takeda, H., Y. Izumi, M. Takahashi, T. Paxton, S. Tamura, T. Koike, Y. Yu, N. Kato, K. Nagase, M. Shiomi, and T. Bamba.

Ameliorating effects of D-47, a newly developed compound, on lipid metabolism in an animal model of familial hypercholesterolemia (WHHLMI rabbits).

Improvements induced in lipid metabolism in the liver by D-47, a newly developed compound, were examined herein. WHHLMI rabbits, an animal model of hypercholesterolemia and coronary atherosclerosis, was fed D-47-supplemented chow for 5 weeks at a dose of 30mg/kg. Lipid concentration were assayed using enzymatic methods. Plasma lipoproteins were fractionated with an ultracentrifuge. mRNA expression was analyzed with real-time PCR. Lipidome analyses of lipoproteins were performed using supercritical fluid chromatography mass spectrometry. In the D-47-treated group, serum lipid levels decreased by 23% for total cholesterol and by 40% for triglycerides. These reductions were mainly attributed to decreases in the VLDL fraction. Compared with the control, in the D-47 group, lipid contents in the liver were decreased by 22% in cholesterol and by 69% in triglycerides, and fat accumulation was decreased by 57% in pericardial fat and by 17% in mesenteric fat. In lipidome analyses of VLDL fraction, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylethanolamine plasmalogen, sphingomyelin, and ceramide were decreased by the D-47 treatment. mRNA expression in the liver was 51% lower for FAS and 24% lower for MTP, but 5.9- and 5.1-fold higher for CYP7A1 and CPT-1, respectively, in the D-47 group than in the control. mRNA expression was 72%, 64%, and 36% higher for LPL, CTP-1, and PPARγ, respectively, in mesenteric fat in the D-47 group. D-47 is a potent lipid-lowering compound that uses a different mechanism of action from that of statins. It has potential as a compound in the treatment of steatohepatitis and metabolic syndrome.

The Coronary Artery Running Pattern is One of the Causes of Individual Differences in the Progression of Coronary Atherosclerosis in WHHLMI Rabbits, an Animal Model for Coronary Atherosclerosis.

AIMS: The relationship between the coronary artery running pattern and development of coronary lesions was re-examined herein using WHHLMI rabbits, an animal model of spontaneous coronary atherosclerosis. METHODS: The coronary artery running pattern was analyzed using an X-ray computed tomography (CT) apparatus after perfusion. Pathological sections were prepared (Victoria blue-HE staining) at 100 µm intervals from the origin of the left circumflex artery (LCX). The severity of coronary lesions was evaluated based on cross-sectional narrowing (lesion area/inner area of the internal elastic lamina). RESULTS: In the CT analysis, the angle of the main curvature of the LCX negatively correlated with the percentage of sections with lesions and cross-sectional narrowing. The percentage of sections with lesions was significantly higher in acute angle-type LCX than in obtuse angle-type LCX. Cross-sectional narrowing was also significantly greater in acute angle-type LCX than in obtuse angle-type LCX. The percentage of fibrous lesions was high at the proximal region of LCX, whereas that of lipid-rich lesions was high at the curvature. In 24 months age group, the percentage of sections with calcification in acute angle-type LCX was about twice that in obtuse angle-type LCX. CONCLUSIONS: Individual differences were observed in the angle of the main curvature of the LCX, which affected the occurrence and extension of atherosclerotic lesions.

Morphological features of coronary plaques in WHHLMI rabbits (Oryctolagus cuniculus), an animal model for familial hypercholesterolemia.

In order to examine their suitability for studies on coronary atherosclerosis, we evaluated the features of coronary atherosclerotic plaques in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, a spontaneous animal model for coronary atherosclerosis and myocardial infarction. Coronary segments of the hearts of 187 WHHLMI rabbits (10-29 months old) were sectioned serially and stained histopathologically and immunohistologically. Progression of coronary lesions was prominent in rabbits that had died suddenly. The degree of coronary lesions of females was higher than that of males. Various types of atherosclerotic lesions were observed in the coronary arteries, such as plaques with a large lipid core covered by a thin fibrous cap, fatty streaks, early and advanced fibroatheromas, fibrous lesions, and advanced lesions with calcium accumulation and the vasa vasorum. In rabbits that had died suddenly, the frequencies of fibroatheromas or advanced lesions were higher than those of rabbits euthanized. Matrix metalloproteinase (MMP)-positive macrophages were detected in gaps among endothelial cells at the plaque surface, beneath the fibrous cap of thin-capped fibroatheromas, and at the bottom of the intimal plaques in which the tunica media was attenuated. Immunohistological results suggest that MMP-positive macrophages are involved in the initiation, progression, and destabilization of coronary plaques, in addition to vascular remodeling, even in WHHLMI rabbits. In conclusion, coronary lesions in WHHLMI rabbits resemble human atherosclerotic lesions, and thus, the WHHLMI rabbit is a suitable animal model for studies on human coronary plaques.

Lipoprotein profiling methodology based on determination of apolipoprotein concentration.

AIM: Abnormal lipid metabolism results in the alteration of lipid compositions in lipoproteins; therefore an accurate and quantitative analytical approach is required for the detailed structural characterization of lipoproteins. However, the specific lipid composition of each lipoprotein particle is poorly understood. MATERIALS & METHODS: Lipid composition of very-low-density lipoprotein and low-density lipoprotein particles derived from myocardial infarction-prone rabbits was determined by normalization of lipidomics data using apoB-100 levels. RESULTS: The ratio of lipid levels between very-low-density lipoprotein and low-density lipoprotein particles was different according to not only lipid classes, but also phosphatidylethanolamine subclasses by applying our developed methodology to myocardial infarction-prone rabbits. CONCLUSION: Our novel analytical approach represents to be a potentially useful tool to obtain particle-specific lipid components of lipoproteins.

High susceptibility of atherosclerotic coronary arteries to the onset of vasospasm and angina pectoris-like symptoms due to coronary spasm in WHHLMI rabbits.

We examined the relationship between atherosclerosis and the provocation of coronary spasm as well as the influence of coronary spasm on the onset of acute ischemic myocardial disease. Coronary spasm was provoked in anesthetized normal Japanese white (JW) rabbits and myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for coronary atherosclerosis and myocardial infarction, by injecting ergonovine during the infusion of norepinephrine through a marginal ear vein. A decrease in contrast flow in the left circumflex artery was observed on coronary angiograms. Ischemic changes were observed on the electrocardiograms of 29% (2/7) of JW and 79% (27/34, P=0.007) of WHHLMI rabbits. The frequency of coronary spasm was significantly high in rabbits with severe coronary plaques showing diffuse lesions. Left ventricle motility in vasospasm-positive rabbits, which was evaluated with echocardiograms, was decreased by 29% following the ergonovine injection (P<0.001), and every serum ischemic marker markedly increased 4 h after the provocation of vasospasm. These results demonstrate that atherosclerotic coronary arteries are positively related to the provocation of vasospasm, and vasospasm in severe atherosclerotic coronary segments evokes angina pectoris-like findings and/or non-fatal myocardial infarction. WHHLMI rabbits may be a novel animal model for angina pectoris and acute ischemic heart disease.

Hyperlipidemia-associated gene variations and expression patterns revealed by whole-genome and transcriptome sequencing of rabbit models.

The rabbit (Oryctolagus cuniculus) is an important experimental animal for studying human diseases, such as hypercholesterolemia and atherosclerosis. Despite this, genetic information and RNA expression profiling of laboratory rabbits are lacking. Here, we characterized the whole-genome variants of three breeds of the most popular experimental rabbits, New Zealand White (NZW), Japanese White (JW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Although the genetic diversity of WHHL rabbits was relatively low, they accumulated a large proportion of high-frequency deleterious mutations due to the small population size. Some of the deleterious mutations were associated with the pathophysiology of WHHL rabbits in addition to the LDLR deficiency. Furthermore, we conducted transcriptome sequencing of different organs of both WHHL and cholesterol-rich diet (Chol)-fed NZW rabbits. We found that gene expression profiles of the two rabbit models were essentially similar in the aorta, even though they exhibited different types of hypercholesterolemia. In contrast, Chol-fed rabbits, but not WHHL rabbits, exhibited pronounced inflammatory responses and abnormal lipid metabolism in the liver. These results provide valuable insights into identifying therapeutic targets of hypercholesterolemia and atherosclerosis with rabbit models.

High-fructose and high-fat diet-induced insulin resistance enhances atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

BACKGROUND: Individuals with insulin resistance and resulting impaired glucose tolerance along with type 2 diabetes showed an increased prevalence of atherosclerosis. Our aim in this study was to address whether diet-induced insulin resistance plays any roles in the development of aortic and coronary atherosclerosis in hyperlipidemic rabbits. METHODS: We fed Watanabe heritable hyperlipidemic (WHHL) rabbits with a high-fructose and high-fat diet (HFFD) with restricted normal calories and compared the lesions of both aortic and coronary atherosclerosis with those of control WHHL rabbits fed a normal chow diet. RESULTS: HFFD-fed WHHL rabbits showed insulin resistance and impaired glucose tolerance accompanied by elevated plasma lipid levels and accumulation of adipose tissue even though their body weight was unchanged compared to the control rabbits. At 8 weeks, the aortic gross lesion area of HFFD-fed WHHL rabbits was increased by 40 % over the controls and their lesions were characterized by increased number of macrophages and smooth muscle cells. At 16 weeks, the lesions of HFFD-fed WHHL rabbits showed more advanced lesions such as lipid core formation and calcification. In addition, coronary atherosclerosis was significantly increased in HFFD-fed WHHL rabbits. CONCLUSIONS: These results suggest that insulin resistance accelerates lesion formation of atherosclerosis.

Lipidomic analysis of plasma lipoprotein fractions in myocardial infarction-prone rabbits.

Lipids play important roles in the body and are transported to various tissues via lipoproteins. It is commonly assumed that alteration of lipid levels in lipoproteins leads to dyslipidemia and serious diseases such as coronary artery disease (CAD). However, lipid compositions in each lipoprotein fraction induced by lipoprotein metabolism are poorly understood. Lipidomics, which involves the comprehensive and quantitative analysis of lipids, is expected to provide valuable information regarding the pathogenic mechanism of CAD. Here, we performed a lipidomic analysis of plasma and its lipoprotein fractions in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. In total, 172 lipids in plasma obtained from normal and WHHLMI rabbits were quantified with high throughput and accuracy using supercritical fluid chromatography hybrid quadrupole-Orbitrap mass spectrometry (SFC/Q-Orbitrap-MS). Plasma levels of each lipid class (i.e., phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine, sphingomyelin, ceramide, triacylglycerol, diacylglycerol, and cholesterol ester, except for free fatty acids) in 21-month-old WHHLMI rabbits were significantly higher than those in normal rabbits. High levels of functional lipids, such as alkyl-phosphatidylcholines, phospholipids including ω-6 fatty acids, and plasmalogens, were also observed in WHHLMI rabbit plasma. In addition, high-resolution lipidomic analysis using very low density lipoprotein (VLDL) and low density lipoprotein (LDL) provided information on the specific molecular species of lipids in each lipoprotein fraction. In particular, higher levels of phosphatidylethanolamine plasmalogens were detected in LDL than in VLDL. Our lipidomics approach for plasma lipoprotein fractions will be useful for in-depth studies on the pathogenesis of CAD.

Probucol suppresses macrophage infiltration and MMP expression in atherosclerotic plaques of WHHL rabbits.

AIM: Probucol is a lipid-lowering drug that is often prescribed for the treatment of familial hypercholesterolemia. However, it is not known whether probucol can change the lesion quality of atherosclerosis. METHODS: We examined this possibility using WHHL rabbits, a model of human familial hypercholesterolemia. Three-month-old male WHHL rabbits were treated with either probucol(85 mg/kg/day) or atorvastatin(6 mg/kg/day) for 16 weeks, and their plasma lipid levels and atherosclerotic lesions were compared with those of a control group. RESULTS: We found that probucol treatment reduced the plasma cholesterol levels, but less remarkably than atorvastatin treatment. In spite of this, probucol treatment led to a prominent reduction of aortic en face lesions by 39%(P＜0.01), whereas atorvastatin reduced these by 16%(P＞0.05), compared with those in the control. Histological examinations revealed that the aortic lesions of probucol-treated rabbits were characterized by reduced macrophages and increased smooth muscle cells compared with those from both the control and atorvastatin groups. Furthermore, probucol treatment reduced the coronary artery stenosis and increased the plaque stability. CONCLUSIONS: These results suggest that probucol treatment may have beneficial effects on the plaque stability of hypercholesterolemic patients.

Contribution of the WHHL rabbit, an animal model of familial hypercholesterolemia, to elucidation of the anti-atherosclerotic effects of statins.

This year marks the 40th year since the discovery of a mutant rabbit showing spontaneous hyperlipidemia, which is the proband of the Watanabe heritable hyperlipidemic (WHHL) rabbit strain, an animal model of familial hypercholesterolemia, and the first statin, a general term for inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase, a rate limiting enzyme in cholesterol biosynthesis. Nowadays, statins are the primary drug of choice for treating cardiovascular disease. Although several reviews have described clinical trials and in vitro studies of statins, the anti-atherosclerotic effects of statins on animal models have not been comprehensively reviewed. This review summarized the contribution of WHHL rabbits to elucidating the anti-atherosclerotic effects of statins in vivo. Studies using WHHL rabbits verified that statins suppress plaque destabilization by reducing unstable components (foam cells derived from macrophages, foam cell debris, and extracellular lipid accumulation), preventing smooth muscle cell reductions, and increasing the collagen content of plaques. In addition, the expression of matrix metalloproteinases and tissue factor are decreased in intimal macrophages by statin treatment. Lipid-lowering effects of statins alter plaque biology by reducing the proliferation and activation of macrophages, a prominent source of the molecules responsible for plaque instability and thrombogenicity. Although statins remain the standard treatment for cardiovascular disease, new therapeutics are eagerly awaited. WHHL rabbits will continue to contribute to the development of therapeutics.

Vasospasm of atherosclerotic coronary arteries precipitates acute ischemic myocardial damage in myocardial infarction-prone strain of the Watanabe heritable hyperlipidemic rabbits.

OBJECTIVE: This study tested the hypothesis that vasospasm can trigger coronary plaque injury and acute ischemic myocardial damage. APPROACH AND RESULTS: Myocardial infarction-prone strain of the Watanabe heritable hyperlipidemic rabbits received an intravenous bolus of ergonovine maleate (0.45 µmol/kg) during intravenous infusion of norepinephrine (12 nmol/kg per minute) to provoke coronary spasm in vivo. After this treatment, coronary angiography demonstrated vasospasm, and the ECG showed ischemic abnormalities (ST depression/elevation and T-wave inversion) in 77% of animals (23/30). These changes normalized after nitroglycerin injection. In rabbits that demonstrated these ECG findings for >20 minutes, echocardiograms showed left ventricular wall motion abnormality. Serum levels of heart-type fatty acid-binding protein, cardiac troponin-I, and myoglobin increased markedly 4 hours after spasm provocation. In coronary lesions of myocardial infarction-prone strain of the Watanabe heritable hyperlipidemic rabbits with provoked coronary spasm, we observed intimal injury in 60.9% in the form of endothelial cell protrusions (39.1%), denudation (30.4%), and macrophage extravasation (56.5%). Plaque disruption with luminal thrombus, however, was only seen in 2 of 23 animals (8.7%), and mural microthrombus was rarely observed (4.3%). CONCLUSIONS: These observations show that provocation of vasospasm in myocardial infarction-prone strain of the Watanabe heritable hyperlipidemic rabbits associates with subsequent ischemic myocardial damage. Although treatment with spasmogens altered aspects of plaque morphology, for example, endothelial protrusion and macrophage emigration, thrombosis was rare in these animals with chronic atherosclerotic disease.

Human apolipoprotein A-II protects against diet-induced atherosclerosis in transgenic rabbits.

OBJECTIVE: Apolipoprotein (apo) A-II is the second major apo of high-density lipoproteins, yet its pathophysiological roles in the development of atherosclerosis remain unknown. We aimed to examine whether apo A-II plays any role in atherogenesis and, if so, to elucidate the mechanism involved. METHODS AND RESULTS: We compared the susceptibility of human apo A-II transgenic (Tg) rabbits to cholesterol diet-induced atherosclerosis with non-Tg littermate rabbits. Tg rabbits developed significantly less aortic and coronary atherosclerosis than their non-Tg littermates, while total plasma cholesterol levels were similar. Atherosclerotic lesions of Tg rabbits were characterized by reduced macrophages and smooth muscle cells, and apo A-II immunoreactive proteins were frequently detected in the lesions. Tg rabbits exhibited low levels of plasma C-reactive protein and blood leukocytes compared with non-Tg rabbits, and high-density lipoproteins of Tg rabbit plasma exerted stronger cholesterol efflux activity and inhibitory effects on the inflammatory cytokine expression by macrophages in vitro than high-density lipoproteins isolated from non-Tg rabbits. In addition, ß-very-low-density lipoproteins of Tg rabbits were less sensitive to copper-induced oxidation than ß-very-low-density lipoproteins of non-Tg rabbits. CONCLUSIONS: These results suggest that enrichment of apo A-II in high-density lipoprotein particles has atheroprotective effects and apo A-II may become a target for the treatment of atherosclerosis.