Watanabe Heritable Hyperlipidemic Rabbits Research Articles

Abstract TP61: Detection of Atherosclerotic Plaque Using Non-contact and Label-free Near-infrared Hyper-spectral Imaging

Background and Aims: Detecting detailed atherosclerotic plaques is important to reduce risk factors during vascular surgery. However, there are few methods to evaluate them during surgery. The aim of this study was to establish an in vivo, non-contact, and label-free imaging method for identifying atherosclerotic plaque lesions from outside vessels with a diffuse-reflectance near-infrared (NIR) hyperspectral imaging (HSI) system. Method: NIR spectra between 1000 and 2350 nm were measured using an NIR HSI imaging system outside the exposed abdominal aorta in 5 Watanabe Heritable Hyperlipidemic (WHHL) rabbits in vivo. Preprocessed data were input to a supervised machine learning algorithm called a support vector machine (SVM) to create pixel-based images that can predict atherosclerotic plaques within a vessel. The images were compared with histological findings. Result: Absorbance was significantly higher in plaques than in normal arteries at 1000-1380, 1580-1810, and 1880-2320 nm. Overall predictive performance showed a sensitivity of 0.814 ± 0.017, a specificity of 0.836 ± 0.020, and an accuracy of 0.827 ± 0.008. The area under the receiver operating characteristic curve was 0.905 (95% confidence interval = 0.904-0.906). Conclusion: The NIR HSI system combined with a machine learning algorithm enabled accurate detection of atherosclerotic plaques within an internal vessel with high spatial resolution from outside the vessel. The findings indicate that the NIR HSI system can provide non-contact, label-free, and precise localization of atherosclerotic plaques during vascular surgery.

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.

In vivo distribution of single chain variable fragment (scFv) against atherothrombotic oxidized LDL/β2-glycoprotein I complexes into atherosclerotic plaques of WHHL rabbits: Implication for clinical PET imaging.

Oxidized LDL (oxLDL) can exist as a complex with β2-glycoprotein I (β2GPI) in plasma/serum of patients with non-autoimmune atherosclerotic disease or antiphospholipid syndrome (APS). Nonetheless, direct in vivo evidence supporting the pathophysiological involvement of oxLDL/β2GPI complexes and specific autoantibody against the complexes in developing atherothrombosis has yet been established. In the present study, we demonstrated in vivo distribution of single chain variable fragment of IgG anti-oxLDL/β2GPI complexes (3H3-scFv) in Watanabe heritable hyperlipidemic (WHHL) rabbits by PET/CT imaging. An antibody-based PET probe, 64Cu-3H3-scFv, was established, and WHHL rabbits were applied for a non-autoimmune atherosclerotic model to demonstrate in vivo distribution of the probe. 3H3-scFv has exhibits specificity towards β2GPI complexed with oxLDL but neither a free form of β2GPI nor oxLDL alone. Post-intravenous administration of 64Cu-3H3-scFv into WHHL rabbits has demonstrated a non-invasive approach for in vivo visualization of atherosclerotic lesion. The imaging probe achieved ideal blood clearance and distribution for optimal imaging capacity in 24h, significantly shorter than that of an intact IgG-based imaging probe. 64Cu-3H3-scFv targeted on atherosclerotic plaques in aortas of WHHL rabbits where extensive accumulation of lipid deposits was observed by lipid staining and autoradiography. The accumulation of 64Cu-3H3-scFv in aortic segments of WHHL rabbits was 2.8-folds higher than that of controls (p=0.0045). The present in vivo evidence supports the pathophysiological involvement of oxLDL/β2GPI complexes in atherosclerotic complications of WHHL rabbits. 64Cu-3H3-scFv represents a novel PET imaging probe for non-invasive pathophysiological assessment of oxLDL/β2GPI complexes accumulated in atherosclerotic plaques.

Structural Remodeling of Sympathetic Innervation in Atherosclerotic Blood Vessels: Role of Atherosclerotic Disease Progression and Chronic Social Stress.

The sympathetic nervous system (SNS) can undergo dramatic structural plasticity in response to behavioral factors and/or the presence of disease, leading to SNS hyperinnervation of peripheral tissues. The SNS has been proposed as an important mediator between stressful behavior and the progression of atherosclerosis in the vasculature. The present study examined whether structural remodeling of the SNS occurs in the vasculature in a genetically hyperlipidemic animal model of atherosclerosis, the Watanabe heritable hyperlipidemic rabbit (WHHL; relative to normolipidemic New Zealand white rabbits [NZW]), and whether SNS plasticity is driven by the progression of disease and/or by stressful social behavior. WHHL and NZW rabbits were assigned to an unstable or stable social environment for 4 months. Aortic atherosclerosis was assessed and SNS aortic innervation quantified using immunofluorescent microscopy. Numerous SNS varicosities were observed throughout the aorta in WHHLs and NZWs, extending into the vascular media and intima, an innervation pattern not previously reported. WHHLs exhibited significantly greater innervation than NZWs (F(1,41) = 55.3, p < .001), with extensive innervation of the atherosclerotic neointima. The innervation density was highly correlated with the extent of disease in the WHHLs (r(21) = 0.855, p < .001). Social environment did not influence innervation in NZWs (aortic arch: p = .078, thoracic aorta: p = .34) or WHHLs (arch: p = .97, thoracic: p = .61). The findings suggest that hyperinnervation is driven largely by the progression of disease rather than social environment. SNS innervation patterns observed in atherosclerotic human and mouse aortas were consistent with the rabbit, suggesting that SNS hyperinnervation of the diseased vessel wall is a general feature across mammalian species.

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.

In vivo detection of atherosclerotic plaque using non-contact and label-free near-infrared hyperspectral imaging

Background and aimsDetecting detailed atherosclerotic plaques is important to reduce risk factors during surgery. However, there are few methods to evaluate them during surgery. The aim of this study was to establish an in vivo, non-contact, and label-free imaging method for identifying atherosclerotic plaque lesions from outside vessels with a diffuse-reflectance near-infrared (NIR) hyperspectral imaging (HSI) system. MethodsNIR spectra between 1000 and 2350 nm were measured using an NIR HSI imaging system outside the exposed abdominal aorta in five Watanabe Heritable Hyperlipidemic (WHHL) rabbits in vivo. Preprocessed data were input to a supervised machine learning algorithm called a support vector machine (SVM) to create pixel-based images that can predict atherosclerotic plaques within a vessel. The images were compared with histological findings. ResultsAbsorbance was significantly higher in plaques than in normal arteries at 1000–1380, 1580–1810, and 1880–2320 nm. Overall predictive performance showed a sensitivity of 0.814 ± 0.017, a specificity of 0.836 ± 0.020, and an accuracy of 0.827 ± 0.008. The area under the receiver operating characteristic curve was 0.905 (95% confidence interval = 0.904–0.906). ConclusionsThe NIR HSI system combined with a machine learning algorithm enabled accurate detection of atherosclerotic plaques within an internal vessel with high spatial resolution from outside the vessel. The findings indicate that the NIR HSI system can provide non-contact, label-free, and precise localization of atherosclerotic plaques during vascular surgery.

Angiotensin II Destabilizes Coronary Plaques in Watanabe Heritable Hyperlipidemic Rabbits.

Increased plasma concentrations of angiotensin II (Ang II) have been implicated in many cardiovascular diseases, such as atherosclerosis, aortic aneurysms, and myocardial infarction, in humans. However, it is not known whether high levels of plasma Ang II affect coronary plaque stability and subsequent myocardial infarction. This study was designed to examine whether elevated plasma Ang II can directly induce coronary events, such as acute coronary syndrome. To examine the above hypothesis, we infused Ang II (100 ng/min per kg [low group] and 200 ng/min per kg [high group]) or saline vehicle via osmotic minipumps into Watanabe heritable hyperlipidemic rabbits, a model of human familial hypercholesterolemia and atherosclerosis. Infusion of Ang II resulted in mortality rates of 50% and 92% in the low- and high-Ang II groups, respectively, whereas there were no deaths in the vehicle group. Pathological analysis revealed that Ang II-infused Watanabe heritable hyperlipidemic rabbits that died showed myocardial infarction. Furthermore, Ang II-infused Watanabe heritable hyperlipidemic rabbits exhibited coronary plaque erosion and rupture that were associated with thrombosis. These findings suggest that increased blood levels of Ang II can destabilize coronary plaques and trigger the thrombosis, which possibly induces myocardial infarction. The model described in this study provides a novel means for the study of human acute coronary syndrome.

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.

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

BackgroundIndividuals 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.MethodsWe 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.ResultsHFFD-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.ConclusionsThese results suggest that insulin resistance accelerates lesion formation of atherosclerosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-015-0024-3) contains supplementary material, which is available to authorized users.

Stabilization of atherosclerotic plaque by pitavastatin in Watanabe heritable hyperlipidemic rabbits: A serial tissue-characterizing intravascular ultrasound study

BackgroundTo examine the effects of pitavastatin on atherosclerotic plaque in Watanabe heritable hyperlipidemic (WHHL) rabbits using serial in vivo tissue-characterizing intravascular ultrasound. MethodsA total of 11 WHHL rabbits of 10–12 weeks of age were divided into two groups, control and pitavastatin-administered groups. A total of 29 atherosclerotic plaque segments from control group and 43 plaque segments from the pitavastatin group were serially imaged by 40MHz intravascular ultrasound in vivo with a tissue characterization software (iMAP™, Boston Scientific, Natick, MA, USA) at the baseline and the follow-up (16th week). ResultsThe level of low-density lipoprotein cholesterol was significantly decreased in pitavastatin group. During the follow-up period, plaque area was significantly increased in the control group, whereas it was not significantly changed in the pitavastatin group. The fibrotic, necrotic, and necrotic plus lipidic areas were significantly increased in the control group, while no significant change was revealed for tissue profile in pitavastatin group. The change in the percent areas of fibrotic and lipidic plus necrotic tissues were significantly different between the two groups especially in the superficial half portion of plaque. ConclusionsThese data indicate that pitavastatin could attenuate atherosclerotic plaque formation and that it could stabilize the plaque in WHHL rabbits. Considering the fact that these were observed even with a high follow-up level of cholesterol, these data might come from the pleiotropic effects of pitavastatin.

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.

MRI study of atherosclerotic plaque progression using ultrasmall superparamagnetic iron oxide in Watanabe heritable hyperlipidemic rabbits.

The purpose of this study was to evaluate plaque progression by using MRI with ultrasmall 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 with 0.8 mmol (Fe) kg(-1) of USPIO (size, 32 nm; concentration, 15 mg dl(-1)). On the fifth post-injection day, they were again given an intravenous injection with 40 μmol kg(-1) 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 analysis of variance [Tukey's honest significant difference (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 (DAKO Corporation, Glostrup, Denmark) and iron uptake in the aortic wall was significantly larger (RAM-11, p < 0.01; iron, p < 0.05). These areas were the smallest in 3-month-old rabbits. Histopathologically, the number of macrophages was the 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. USPIO-enhanced MRI visualized the accumulation of macrophages in early atherosclerotic plaques of WHHL rabbits in the course of natural progression.

The Arginine/ADMA Ratio Is Related to the Prevention of Atherosclerotic Plaques in Hypercholesterolemic Rabbits When Giving a Combined Therapy with Atorvastatine and Arginine.

Supplementation with arginine in combination with atorvastatin is more efficient in reducing the size of an atherosclerotic plaque than treatment with a statin or arginine alone in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits. We evaluated the mechanism behind this feature by exploring the role of the arginine/asymmetric dimethylarginine (ADMA) ratio, which is the substrate and inhibitor of nitric oxide synthase (NOS) and thereby nitric oxide (NO), respectively. Methods: Rabbits were fed either an arginine diet (group A, n = 9), standard rabbit chow plus atorvastatin (group S, n = 8), standard rabbit chow plus an arginine diet with atorvastatin (group SA, n = 8) or standard rabbit chow (group C, n = 9) as control. Blood was sampled and the aorta was harvested for topographic and histological analysis. Plasma levels of arginine, ADMA, cholesterol and nitric oxide were determined and the arginine/ADMA ratio was calculated. Results: The decrease in ADMA levels over time was significantly correlated to fewer aortic lesions in the distal aorta and total aorta. The arginine/ADMA ratio was correlated to cholesterol levels and decrease in cholesterol levels over time in the SA group. A lower arginine/ADMA ratio was significantly correlated to lower NO levels in the S and C group. Discussion: A balance between arginine and ADMA is an important indicator in the prevention of the development of atherosclerotic plaques.

Compensatory UTE/T2W Imaging of Inflammatory Vascular Wall in Hyperlipidemic Rabbits.

ObjectivesTo obtain compensatory ultra-short echo time (UTE) imaging and T2-weighted (T2W) imaging of Watanabe heritable hyperlipidemic (WHHL) rabbits following dextran-coated magnetic nanocluster (DMNC) injection for the effective in vivo detection of inflammatory vascular wall.MethodsMagnetic nanoparticle was synthesized by thermal decomposition and encapsulated with dextran to prepare DMNC. The contrast enhancement efficiency of DMNC was investigated using UTE (repetition time [TR] = 5.58 and TE = 0.07 ms) and T2W (TR = 4000 and TE = 60 ms) imaging sequences. To confirm the internalization of DMNC into macrophages, DMNC-treated macrophages were visualized by cellular transmission electron microscope (TEM) and magnetic resonance (MR) imaging. WHHL rabbits expressing macrophage-rich plaques were subjected to UTE and T2W imaging before and after intravenous DMNC (120 μmol Fe/kg) treatment. Ex vivo MR imaging of plaques and immunostaining studies were also performed.ResultsPositive and negative contrast enhancement of DMNC solutions with increasing Fe concentrations were observed in UTE and T2W imaging, respectively. The relative signal intensities of the DMNC solution containing 2.9 mM Fe were calculated as 3.53 and 0.99 in UTE and T2W imaging, respectively. DMNC uptake into the macrophage cytoplasm was visualized by electron microscopy. Cellular MR imaging of DMNC-treated macrophages revealed relative signals of 3.00 in UTE imaging and 0.98 in T2W imaging. In vivo MR images revealed significant brightening and darkening of plaque areas in the WHHL rabbit 24 h after DMNC injection in UTE and T2W imaging, respectively. Ex vivo MR imaging results agreed with these in vivo MR imaging results. Histological analysis showed that DMNCs were localized to areas of inflammatory vascular wall.ConclusionsUsing compensatory UTE and T2W imaging in conjunction with DMNC is an effective approach for the noninvasive in vivo imaging of atherosclerotic plaque.

Irbesartan attenuates atherosclerosis in Watanabe heritable hyperlipidemic rabbits: noninvasive imaging of inflammation by 18F-fluorodeoxyglucose positron emission tomography.

The purpose of this study was to assess the usefulness of 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) in evaluating the antiatherogenic effects of irbesartan, an angiotensin II type 1 receptor blocker. Watanabe heritable hyperlipidemic rabbits were divided into the irbesartan-treated group (75 mg/kg/d; n = 14) and the control group (n = 14). After a 9-month treatment, rabbits underwent 18F-FDG PET. Using the aortic lesions, autoradiography and histologic examinations were performed. PET imaging clearly visualized the thoracic lesions of control rabbits and showed a significant decrease in the 18F-FDG uptake level of irbesartan-treated rabbits (78.8% of controls; p < .05). Irbesartan treatment significantly reduced the plaque size (43.1% of controls) and intraplaque macrophage infiltration level (48.1% of controls). The 18F-FDG uptake level in plaques positively correlated with the plaque size (r = .65, p < .05) and macrophage infiltration level (r = .57, p < .05). Noninvasive imaging by 18F-FDG PET is useful for evaluating the therapeutic effects of irbesartan and reflects inflammation, a key factor involved in the therapeutic effects.

Abstract 50: A Phase I Clinical Trial Evaluating the Safety of With Allogeneic Adipose Tissue-derived Multilineage Progenitor Cells-transplantation Therapy in Homozygous Familial Hypercholesterolemia Patients

Familial hypercholesterolemia (FH) is an inherited disorder, mainly caused by defects in low-density lipoprotein (LDL) receptor gene. The patients are characterized by high LDL cholesterol levels in the blood and premature cardiovascular disease. Although most of heterozygous FH patients are usually treated with statin, ezetimibe and bile acid sequestrants, homozygous FH patients are resistant to drug therapy. Therefore, in Japan, many of homozygous FH patients are treated by LDL-apheresis. LDL-apheresis is a great procedure to remove LDL cholesterol from the blood and contribute to improve prognosis of homozygous FH patients. However, the effect of removing LDL cholesterol is temporary and still not enough. As a definitive therapy, liver transplantation therapy could be one of options to recover LDL receptor, but donor is limited in Japan. Therefore, based on the increase of the evidence about the safety of mesenchymal stem cells and percutaneous transhepatic portal approach in islet transplantation, we have developed a cell transplantation therapy with allogeneic adipose tissue-derived multilineage progenitor cells (ADMPCs), as an alternative treatment instead of liver transplantation. Our group has already proved that xenogenic transplantation of human ADMPCs into Watanabe heritable hyperlipidemic rabbits resulted in significant reductions in total cholesterol, and the reductions were observed within 4 weeks and maintained for 12 weeks. These results suggested that hADMPC transplantation could correct the metabolic defects and be a novel therapy for inherited liver diseases. Here, we report a protocol for the first-in-human clinical trial, which has been approved by the institutional review board and Ministry of Health, Labour and Welfare, Japan.

Bisphenol A exposure induces metabolic disorders and enhances atherosclerosis in hyperlipidemic rabbits

Bisphenol A (BPA) is an artificial environmental endocrine disrupter. Excess exposure to BPA may induce many disorders in the metabolism and cardiovascular system. However, the underlying toxicological mechanisms remain largely unknown. In this study, we administered genetically hyperlipidemic Watanabe heritable hyperlipidemic (WHHL-MI) rabbits (male, 14 week old), which have more common features with humans than the mouse and rat especially in the metabolism and cardiovascular system, with BPA at 40 mg kg(-1) day(-1) for 8 weeks by gavage and compared their plasma lipids, glucose and insulin response with those of the vehicle group. All of the rabbits were sacrificed, and their pancreas, liver, adipose tissue, heart and aorta were analyzed using histological and morphometric methods. Furthermore, we treated human hepatoma HepG2 cells and human umbilical cord vein endothelial cells (HUVECs), with different doses of BPA based on the serum BPA levels in the WHHL rabbits for 6 h to investigate the possible molecular mechanisms. Our results showed that BPA-treated rabbits showed insulin resistance, prominent adipose accumulation and hepatic steatosis. Additionally, BPA exposure also caused myocardial injury and enhanced the development of atherosclerosis in the aortic arch with increased macrophage number (86%) and advanced lesion areas (69%). Increased expression of inflammatory genes found in the liver of BPA-treated rabbits along with the up-regulation of ER stress, lipid and glucose homeostasis and inflammatory genes in the cultured HepG2 cells and HUVECs suggest that BPA may induce metabolic disorders and enhance atherosclerosis through regulating above molecular pathways in the liver and endothelium.

Comparison of 64Cu and 68Ga for Molecular Imaging of Atherosclerosis using the Apolipoprotein A-I Mimetic Peptide FAMP

Background: Molecular imaging for detection of the atherosclerotic plaque burden has been highlighted as a modality for the diagnosis of atherosclerosis. We recently developed a novel and noninvasive positron emission tomography (PET) that was functionalized with an apolipoprotein (Apo) A-I mimetic peptide [known as Fukuoka University Apo A-I mimetic peptide (FAMP)] radiolabeled with gallium-68 (68Ga) - 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) to specifically image the status of atherosclerotic plaque in myocardial infarctionprone Watanabe heritable hyperlipidemic rabbits (WHHL-MI). Methods and Results: To achieve more sensitive molecular imaging, FAMP was modified with 4, 11 - bis (carboxymethyl) - 1, 4, 8, 11 - tetraazabicyclo (6.6.2) hexadecane (CB-TE2A) and radiolabeled with copper-64 (64Cu) for PET, and the ability of 64Cu-TE2A-FAMP to image plaque was compared with that of 68Ga-DOTA-FAMP. Japanese white normal (JW) and WHHL-MI rabbits were intravenously injected with 64Cu-CB-TE2A-FAMP or 68Ga- DOTA-FAMP, and subjected to continuous PET (25-30 MBq). Interestingly, 64Cu-CB-TE2A-FAMP was not taken up by atherosclerotic lesions in the aorta of WHHL-MI, whereas 68Ga-DOTA-FAMP was dramatically illuminated in the aorta of WHHL-MI. Moreover, 64Cu-CB-TE2A-FAMP was rapidly decomposed and 64Cu was excreted to the intestine, liver or urinary bladder in both JW and WHHL-MI rabbits. Conclusions: These results demonstrated that FAMP may be a target molecule for atherosclerotic molecular imaging with 68Ga-DOTA, but not with 64Cu-CB-TE2A. The selection of a suitable radio-nuclide and chelator might be important for HDL functioning imaging.

Bisphenol A exposure enhances atherosclerosis in WHHL rabbits.

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.

Rabbit models for the study of human atherosclerosis: from pathophysiological mechanisms to translational medicine.

Laboratory animal models play an important role in the study of human diseases. Using appropriate animals is critical not only for basic research but also for the development of therapeutics and diagnostic tools. Rabbits are widely used for the study of human atherosclerosis. Because rabbits have a unique feature of lipoprotein metabolism (like humans but unlike rodents) and are sensitive to a cholesterol diet, rabbit models have not only provided many insights into the pathogenesis and development of human atherosclerosis but also made a great contribution to translational research. In fact, rabbit was the first animal model used for studying human atherosclerosis, more than a century ago. Currently, three types of rabbit model are commonly used for the study of human atherosclerosis and lipid metabolism: (1) cholesterol-fed rabbits, (2) Watanabe heritable hyperlipidemic rabbits, analogous to human familial hypercholesterolemia due to genetic deficiency of LDL receptors, and (3) genetically modified (transgenic and knock-out) rabbits. Despite their importance, compared with the mouse, the most widely used laboratory animal model nowadays, the use of rabbit models is still limited. In this review, we focus on the features of rabbit lipoprotein metabolism and pathology of atherosclerotic lesions that make it the optimal model for human atherosclerotic disease, especially for the translational medicine. For the sake of clarity, the review is not an attempt to be completely inclusive, but instead attempts to summarize substantial information concisely and provide a guideline for experiments using rabbits.