[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
These results suggest that insulin resistance accelerates lesion formation of atherosclerosis.
Full-text · Article · Dec 2015 · Nutrition & Metabolism
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jul 2015 · Journal of Bioscience and Bioengineering
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to evaluate plaque progression by MRI using ultra-small superparamagnetic iron oxide (USPIO) and by histopathological studies.
We divided 12 Watanabe heritable hyperlipidemic (WHHL) rabbits into 4 groups based on their age (3-, 9-, 14-, and 26 months) and injected them intravenously (i.v.) with 0.8 mmol iron (Fe)/kg of USPIO (size 32 nm, concentration 15mg/dl). On the 5th post-injection day they were again given an i.v. injection with 40 μmol/kg of the same USPIO and MR angiography (MRA) was performed. The signal-to-noise ratio (SNR) in regions of interest in the wall of the upper abdominal aorta was calculated on coronal images. Specimens from the same level of the aorta were subjected to iron-staining and RAM-11 immunostaining and used for histopathological study. For statistical analysis of the MRA- and histopathological findings we used ANOVA (Tukey's HSD test).
In 9-month-old rabbits the SNR was significantly lower than in rabbits of the other ages (p < 0.01) and the area of RAM-11- and iron uptake in the aortic wall was significantly larger (RAM-11, p < 0.01; iron, p < 0.05). These areas were smallest in 3-month-old rabbits.
Histopathologically, the number of macrophages was greatest in 9-month-old rabbits. Our findings indicate that the SNR on MRI scans reflects the number of macrophages in the aortic wall of WHHL rabbits. Advances in knowledge: USPIO-enhanced MRI visualized the accumulation of macrophages in the early atherosclerotic plaques of WHHL rabbits in the course of natural progression.
No preview · Article · Jun 2015 · The British journal of radiology
[Show abstract][Hide abstract] ABSTRACT: Bisphenol A (BPA) is an environmental endocrine disrupter. Excess exposure to BPA may increase susceptibility to many metabolic disorders, but it is unclear whether BPA exposure has any adverse effects on the development of atherosclerosis. To determine whether there are such effects, we investigated the response of Watanabe heritable hyperlipidemic (WHHL) rabbits to 400-µg/kg BPA per day, administered orally by gavage, over the course of 12 weeks and compared aortic and coronary atherosclerosis in these rabbits to the vehicle group using histological and morphometric methods. In addition, serum BPA, cytokines levels and plasma lipids as well as pathologic changes in liver, adipose and heart were analyzed. Moreover, we treated human umbilical cord vein endothelial cells (HUVECs) and rabbit aortic smooth muscle cells (SMCs) with different doses of BPA to investigate the underlying molecular mechanisms involved in BPA action(s). BPA treatment did not change the plasma lipids and body weights of the WHHL rabbits; however, the gross atherosclerotic lesion area in the aortic arch was increased by 57% compared to the vehicle group. Histological and immunohistochemical analyses revealed marked increases in advanced lesions (37%) accompanied by smooth muscle cells (60%) but no significant changes in the numbers of macrophages. With regard to coronary atherosclerosis, incidents of coronary stenosis increased by 11% and smooth muscle cells increased by 73% compared to the vehicle group. Furthermore, BPA-treated WHHL rabbits showed increased adipose accumulation and hepatic and myocardial injuries accompanied by up-regulation of endoplasmic reticulum (ER) stress and inflammatory and lipid metabolism markers in livers. Treatment with BPA also induced the expression of ER stress and inflammation related genes in cultured HUVECs. These results demonstrate for the first time that BPA exposure may increase susceptibility to atherosclerosis in WHHL rabbits.
[Show abstract][Hide abstract] ABSTRACT: Increased plasma levels of C-reactive protein (CRP) are closely associated with cardiovascular diseases, but whether CRP is directly involved in the pathogenesis of atherosclerosis is still under debate. Many controversial and contradictory results using transgenic mice and rabbits have been published but it is also unclear whether CRP lowering can be used for the treatment of atherosclerosis. In the current study, we examined the effects of the rabbit CRP antisense oligonucleotides (ASO) on the development of atherosclerosis in WHHL rabbits. CRP ASO treatment led to a significant reduction of plasma CRP levels; however, both aortic and coronary atherosclerotic lesions were not significantly changed compared to those of control WHHL rabbits. These results suggest that inhibition of plasma CRP does not affect the development of atherosclerosis in WHHL rabbits.
Full-text · Article · Apr 2014 · Mediators of Inflammation
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
These results suggest that probucol treatment may have beneficial effects on the plaque stability of hypercholesterolemic patients.
No preview · Article · Mar 2014 · Journal of atherosclerosis and thrombosis
[Show abstract][Hide abstract] ABSTRACT: In this study we present a novel image analysis methodology to quantify and to classify morphological details in tissue collagen fibril organization and lipid deposition. Co-localized collagen (second harmonic, SHG) and lipid (coherent Raman, CARS) images of atherosclerotic artery walls were acquired by a supercontinuum-powered multi-modal nonlinear microscope. Textural features based on the first-order statistics (FOS) and gray level co-occurrence matrix (GLCM) parameters were extracted from the SHG and CARS images. Multi-group classifications based on support vector machine of SHG and CARS images were subsequently performed to investigate the potential of texture analysis in providing quantitative descriptors of structural and compositional changes during disease progression. Using a rabbit model, different collagen remodeling and lipid accumulation patterns in disease tissues can be successfully tracked using these image statistics, thus providing a robust foundation for classification. When the variation of the CARS image features were tracked against the age of the rabbit, it was noticed that older animals (advanced plaques) present a more complex necrotic core containing high-lipid extracellular structures with various shapes and distribution. With combined FOS and GLCM texture statistics, we achieved reliable classification of SHG and CARS images acquired from atherosclerotic arteries with >90% accuracy, sensitivity and specificity. The proposed image analysis methodology can also be applied in a wide range of applications to evaluate conditions involving collagen re-modeling and prominent lipid accumulation.
No preview · Article · Jan 2014 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: We used magnetic resonance imaging (MRI) and histologic techniques to compare the uptake by the rabbit atherosclerotic wall of 4 types of superparamagnetic iron oxide (SPIO) particles, i.e. SPIO, mannan-coated SPIO (M-SPIO), ultrasmall SPIO (USPIO), and mannan-coated USPIO (M-USPIO).
All experimental protocols were approved by our institutional animal experimentation committee. We intravenously injected 12 Watanabe heritable hyperlipidemic rabbits with one of the 4 types of SPIO (0.8mmol Fe/kg). Two other rabbits served as the control. The rabbits underwent in vivo contrast-enhanced magnetic resonance angiography (MRA) before- and 5 days after these injections; excised aortae were subjected to in vitro MRI. In the in vivo and in vitro studies we assessed the signal intensity of the vessels at identical regions of interest (ROI) and calculated the signal-to-noise ratio (SNR). For histologic assessment we evaluated the iron-positive regions in Prussian blue-stained specimens.
There were significant differences in iron-positive regions where M-USPIO>USPIO, M-SPIO>SPIO, USPIO>SPIO (p<0.05) but not between M-USPIO and M-SPIO. The difference between the pre- and post-injection SNR was significantly greater in rabbits treated with M-USPIO than USPIO and in rabbits injected with M-SPIO than SPIO (p<0.05). On in vitro MRI scans SNR tended to be lower in M-USPIO- and M-SPIO- than USPIO- and SPIO-treated rabbits (p<0.1).
Histologic and imaging analysis showed that mannan-coated SPIO and USPIO particles were taken up more readily by the atherosclerotic rabbit wall than uncoated SPIO and USPIO.
No preview · Article · Aug 2013 · European journal of radiology
[Show abstract][Hide abstract] ABSTRACT: This study tested the hypothesis that vasospasm can trigger coronary plaque injury and acute ischemic myocardial damage.
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%).
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.
Preview · Article · Aug 2013 · Arteriosclerosis Thrombosis and Vascular Biology
[Show abstract][Hide abstract] ABSTRACT: In this study we present an image analysis methodology capable of quantifying morphological changes in tissue collagen fibril organization caused by pathological conditions. Texture analysis based on first-order statistics (FOS) and second-order statistics such as gray level co-occurrence matrix (GLCM) was explored to extract second-harmonic generation (SHG) image features that are associated with the structural and biochemical changes of tissue collagen networks. Based on these extracted quantitative parameters, multi-group classification of SHG images was performed. With combined FOS and GLCM texture values, we achieved reliable classification of SHG collagen images acquired from atherosclerosis arteries with >90% accuracy, sensitivity and specificity. The proposed methodology can be applied to a wide range of conditions involving collagen re-modeling, such as in skin disorders, different types of fibrosis and muscular-skeletal diseases affecting ligaments and cartilage.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Erythropoietin (EPO), a type I cytokine originally identified for its critical role in hematopoiesis, has been shown to have nonhematopoietic, tissue-protective effects, including suppression of atherosclerosis. However, prothrombotic effects of EPO hinder its potential clinical use in non-anemic patients. METHODS AND RESULTS: In the present study, we investigated the anti-atherosclerotic effects of helix B surface peptide (HBSP), a nonerythropoietic, tissue-protective compound derived from EPO, using human umbilical vein endothelial cells (HUVECs) and human monocytic THP-1 cells in vitro and Watanabe heritable hyperlipidemic spontaneous myocardial infarction (WHHLMI) rabbits in vivo. In HUVECs, HBSP inhibited apoptosis (≈70%) induced by C-reactive protein (CRP), a direct mediator of atherosclerosis. Using a small interfering RNA approach, Akt was shown to be a key molecule in HBSP-mediated prevention of apoptosis. HBSP also attenuated CRP-induced production of TNF-α and matrix metalloproteinase-9 in THP-1 cells. In the WHHLMI rabbit, HBSP significantly suppressed progression of coronary atherosclerotic lesions as assessed by mean cross-sectional stenosis (HBSP; 21.3 ± 2.2% vs. control peptide; 38.0 ± 2.7%) and inhibited coronary artery endothelial cell apoptosis with increased activation of Akt. Furthermore, TNF-α expression and the number of M1 macrophages and M1/M2 macrophage ratio in coronary atherosclerotic lesions were markedly reduced in HBSP-treated animals. CONCLUSIONS: These data demonstrate that HBSP suppresses coronary atherosclerosis, in part by inhibiting endothelial cell apoptosis through activation of Akt and in association with decreased TNF-α production and modified macrophage polarization in coronary atherosclerotic lesions. Since HBSP does not have the prothrombotic effects of EPO, our study may provide a novel therapeutic strategy that prevents progression of coronary artery disease.
Full-text · Article · May 2013 · Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: Background:
High-density lipoprotein (HDL) plays a major role in reverse cholesterol transport. Many researchers have been working to enhance the biochemical function of HDL for use in therapy. Although HDL therapy using injections of apolipoprotein (apo)-A-I mimetics, apo A-I Milano or full-length apo A-I is dramatically effective, it is still unclear whether apo A-I or apo A-I mimetics actually enter atherosclerotic plaque and remove cholesterol from the lipid burden. We synthesized a novel 24-amino acid apo A-I mimetic peptide (known as FAMP) that potently removes cholesterol via specific ATP-binding cassette transporter A1. We then investigated the potential of FAMP to image developing plaque lesions in vivo.
Methods and results:
FAMP was modified with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and radiolabeled with gallium-68 ((68)Ga) for noninvasive positron emission tomography (PET) in an animal model (familial hypercholesterolemic myocardial infarction-prone rabbits: WHHL-MI) with atherosclerotic lesions. The (68)Ga-DOTA-FAMP was dramatically taken up by atherosclerotic tissues in the blood vessels and aorta of WHHL-MI rabbits, but not the control rabbits.
An apo A-I mimetic peptide with (68)Ga-DOTA is a promising candidate diagnostic tracer for PET imaging of the atherosclerotic lipid burden and may contribute to the development of a tool for the diagnosis of plaque with PET.
Full-text · Article · Mar 2013 · Circulation Journal
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Despite the significant effort in developing radioprobes for atherosclerosis, few have low molecular weight. Oxidized LDL (OxLDL), a highly proinflammatory and proatherogenic factor that is abundant in atherosclerotic plaques, plays a pivotal role in plaque destabilization, which makes OxLDL a relevant probe target. We developed a radioiodinated short peptide, AHP7, as a low molecular weight probe for specific OxLDL imaging and evaluated its utility using myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits (WHHLMI).
[¹²⁵I]AHP7 was designed and synthesized based on the sequence of Asp-hemolysin, an OxLDL binding protein extracted from Aspergillus fumigatus. In vitro binding studies with OxLDL having varying degrees of oxidation were performed. Radioactivity accumulation in the aorta was measured 30 min post-administration in rabbits. Autoradiography and histological studies were performed using serial aorta sections. A radioiodinated scrambled peptide ([¹²⁵I]AHP scramble) was used as a negative control.
[¹²⁵I]AHP7 bound to OxLDL in proportion to the degree of oxidation (R=0.91, P<0.0001) and was inhibited by unlabeled AHP7 in a concentration-dependent manner. The aorta accumulation level and aorta/blood and aorta/muscle ratios of [¹²⁵I]AHP7 in WHHLMI were 2.8-, 1.3- and 1.8-fold higher, respectively, than those in control rabbits (P<0.001). Co-administration of AHP7 significantly reduced [¹²⁵I]AHP7 radioactivity in aorta sections (P<0.0001). Regional radioactivity levels in the aorta sections showed nonuniformity but similarity to the immunohistochemical OxLDL density.
The potential of radioiodinated AHP7 for selectively imaging OxLDL was demonstrated both in vitro and in vivo.
No preview · Article · Nov 2012 · Nuclear Medicine and Biology
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine whether features indicative of myocardial ischemia occur in the electrocardiograms (ECG) in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for human familial hypercholesterolemia. ECG were recorded in 110 anesthetized WHHLMI rabbits (age, 10 to 39 mo) by using unipolar and bipolar limb leads with or without chest leads. We noted the following electrocardiographic changes: T wave inversion (37.4%), ST segment depression (31.8%), deep Q wave (16.3%), reduced R wave amplitude (7.3%), ST segment elevation (2.7%), and high T wave (1.8%). These ECG changes resembled those in human patients with coronary heart disease. Histopathologic examination revealed that the left ventricular wall showed acute myocardial lesions, including loss of cross-striations, vacuolar degeneration, coagulation necrosis of cardiac myocytes, and edema between myofibrils, in addition to chronic myocardial lesions such as myocardial fibrosis. The coronary arteries that caused these ECG changes were severely stenosed due to atherosclerotic lesions. Ischemic ECG changes corresponded to the locations of the myocardial lesions. Normal ECG waveforms were similar between WHHLMI rabbits and humans, in contrast to the large differences between rabbits and mice or rats. In conclusion, ischemic ECG changes in WHHLMI rabbits reflect the location of myocardial lesions, making this model useful for studying coronary heart disease.
Preview · Article · Oct 2012 · Comparative medicine
[Show abstract][Hide abstract] ABSTRACT: To examine whether the myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbit with visceral fat accumulation is a new animal model for human metabolic syndrome, we examined the relationship between mesenteric fat accumulation and insulin resistance, hyperlipidemia and atherosclerosis.
Glucose tolerance tests were performed using adult (11- to 15-month-old) and middle-aged (17- to 21-month-old) WHHLMI rabbits fed standard chow restrictedly. In addition, lipoprotein lipid levels, serum C-reactive protein (CRP) levels, mesenteric fat weight and physical and physiological parameters were measured. Mesenteric fat was stained immunohistochemically.
The mesenteric adipose tissue was positive for monoclonal antibodies against macrophages, C-reactive protein and monocyte chemoattractant protein. In adult rabbits, mesenteric fat correlated to aortic lesion area, insulin resistance, fasting immunoreactive insulin, serum CRP, abdominal circumference and body weight. In middle-aged rabbits, mesenteric fat correlated to lipoprotein lipid levels in addition to the parameters showing a significant correlation in adult rabbits, excluding aortic lesion area.
The WHHLMI rabbit with visceral fat accumulation is a new animal model for metabolic syndrome.