Watanabe Heritable Hyperlipidemic Rabbits Research Articles

Arterial stiffness of the aorta and iliofemoral artery and their responses to nitroglycerin administration in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits.

The role of hypercholesterolemia in arterial stiffness, which usually reflects the progression of atherosclerosis has not been fully investigated. To clarify the meaning of arterial stiffness in hypercholesterolemia, we evaluated arterial stiffness in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits by using new arterial stiffness indices of the aorta and common iliac to femoral artery. The new arterial stiffness indices of both arteries were determined by the application of the theory of cardio-ankle vascular index (CAVI) to the aorta (aBeta) and ilio-femoral artery (ifBeta). Furthermore, the responses of both indices to nitroglycerin (NTG) administration were compared between WHHHMI and normal rabbits. aBeta and ifBeta of WHHLMI and normal rabbits were measured under anesthesia. Pulse wave velocity in the whole aorta (aPWV) and ilio-femoral artery (ifPWV), blood pressure, and other parameters were measured before and after administration of NTG (50-120 μg/kg/min) every 1 for 5 min. Atherosclerotic lesions were observed in the aorta, but a little in the ilio-femoral artery in WHHLMI rabbits. Compared with normal rabbits, aBeta was significantly higher, but ifBeta was lower in WHHLMI rabbits. When NTG was administered, ifBeta decreased significantly in both groups; however, aBeta increased in normal rabbits, but remained unchanged in WHHIMI rabbits. These findings suggested that hereditary hypercholesterolemia in rabbits did not uniformly enhance arterial stiffness in elastic artery and muscular artery. The responses to NTG were also different between two arteries. The mechanism of these different responses needs further studies.

Development of an image classification pipeline for atherosclerotic plaques assessment using supervised machine learning

BackgroundDuring atherosclerosis, the narrowing of the arterial lumen is observed through the accumulation of bio compounds and the formation of plaque within artery walls. A non-linear optical imaging modality (NLOM), coherent anti-stokes Raman scattering (CARS) microscopy, can be used to image lipid-rich structures commonly found in atherosclerotic plaques. By matching the lipid’s molecular vibrational frequencies (CH bonds), it is possible to map the accumulation of lipid-rich structures without the need for exogenous labelling and/or processing of the samples. CARS allows for the visualization of the morphological features of plaque. In combination with supervised machine learning, CARS imaged morphological features can be used to characterize the progression of atherosclerotic plaques. ResultsBased on a set of label-free CARS images of atherosclerotic plaques (i.e. foam cell clusters) from a Watanabe heritable hyperlipidemic rabbit model, we developed an automated pipeline to classify atherosclerotic lesions based on their major morphological features. Our method uses image preprocessing to first improve the quality of the CARS-imaged plaque, followed by the segmentation of the plaque using Otsu thresholding, marker-controlled watershed, K-means segmentation and a novel independent foam cell thresholding segmentation. To define relevant morphological features, 27 quantitative features were extracted and further refined by a novel coefficient of variation feature refinement method in accordance with filter-type feature selection. Refined morphological features were supplied into three supervised machine learning algorithms; K-nearest neighbour, support vector machine and decision tree classifier. The classification pipeline showcased the ability to exploit relevant plaque morphological features to accurately classify 3 pre-defined stages of atherosclerosis: early fatty streak development (EFS) and advancing atheroma (AA) with a greater than 85% class accuracyConclusionsThrough the combination of CARS microscopy and computational methods, a powerful classification tool was developed to identify the progression of atherosclerotic plaque in an automated manner. Using a curated dataset, the classification pipeline demonstrated the ability to differentiate between EFS, EF and AA. Thus, presenting the opportunity to classify the onset of atherosclerosis at an earlier stage of development

Pathological Investigations of Intracranial Atherosclerosis Using Multiple Hypercholesterolemic Rabbit Models.

BackgroundIntracranial atherosclerosis (ICAS) is one of the most common causes of ischemic stroke, but there are few animal models that can recapitulate its pathological features. In this study, we examined ICAS pathological features and anatomic distributions using three types of hyperlipidemic rabbit models. We also investigated the effect of different lipoprotein profiles and hypertension on ICAS.Materials and MethodsWe examined Watanabe heritable hyperlipidemic (WHHL) rabbits, apoE knockout (KO) rabbits and wild-type rabbits (WT) fed a cholesterol diet, in addition to WT rabbits fed a standard diet as a control. The whole brain was dissected and embedded in paraffin. Serial sections were stained with either hematoxylin/eosin or elastica van Gieson, or immunohistochemically stained with monoclonal antibodies against macrophages and smooth muscle cells. We investigated (1) the presence of cerebral atherosclerosis; (2) the lesion locations in the cerebral arteries; (3) the degree of lumen stenosis; (4) pathological features and cellular components of the lesions in these rabbits; and (5) whether hypertension affects ICAS.ResultsICAS was detected in apoE and WHHL rabbits, but not in WT rabbits. Compared with apoE KO rabbits, WHHL rabbits had greater ICAS. The lesions of cerebral atherosclerosis were mainly distributed at the bifurcations of the posterior cerebral artery, basilar artery and vertebral artery, and they were basically characterized by smooth muscle cells and extracellular matrix with few macrophages. The extent of the ICAS in WHHL rabbits was significantly increased by hypertension.ConclusionsICAS was detected in WHHL and apoE KO rabbits, and occurred in specific locations in the cerebral arteries. Hypertension promotes the development of ICAS in the setting of hypercholesterolemia.

Effects of chronic mirabegron treatment on metabolic and cardiovascular parameters as well as on atherosclerotic lesions of WHHL rabbits with high-fructose high-fat diet-induced insulin resistance.

Background and aimMetabolic syndrome (MetS) is a global health and economic burden. Finding a suitable pharmacological approach for managing this syndrome is crucial. We explored the therapeutic potential of mirabegron (MIR), a β3-adrenergic receptor agonist, as a repurposed agent for the treatment of MetS and its cardiovascular consequences. MethodsThirty Watanabe heritable hyperlipidemic rabbits (WHHL) were divided into 3 groups: control, high-fructose high-fat diet (HFFD) and HFFD + MIR that received a chow diet, HFFD and HFFD along with MIR treatment, respectively. The protocol lasted for 12 weeks, during which weight and abdominal circumference were monitored; plasma fasting levels of lipids, glucose and insulin were measured and an intravenous glucose tolerance test (IVGTT) was performed. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Cardiac function was assessed using in-vivo and ex-vivo approaches. Vascular reactivity was estimated via isolated carotid arteries method. Aortic atherosclerosis was evaluated using histological and immuno-histochemical techniques. ResultsIn contrast to the HFFD group, MIR-treated rabbits showed fasting insulin, HOMA-IR and TG levels stabilization and exhibited improved cardiac inotropy and lusitropy, while on the other hand, displayed aggravated atheroma plaque development. ConclusionLong-term treatment with MIR prevented the increase in TG levels and the establishment of IR and enhanced the cardiac function of a rabbit animal model of MetS with combined dyslipidemia and IR.

Long-term high-fructose high-fat diet feeding elicits insulin resistance, exacerbates dyslipidemia and induces gut microbiota dysbiosis in WHHL rabbits.

The metabolic syndrome (MetS) has become a global public health burden due to its link to cardiovascular disease and diabetes mellitus. The present study was designed to characterize the metabolic and cardiovascular disturbances, as well as changes in gut microbiota associated with high-fructose high-fat diet (HFFD)-induced MetS in Watanabe heritable hyperlipidemic (WHHL) rabbits. Twenty-one Watanabe rabbits were assigned to a control (n = 9) and HFFD (n = 12) groups, receiving a chow diet and a HFFD, respectively. During a 12-weeks protocol, morphological parameters were monitored; plasma fasting levels of lipids, glucose and insulin were measured and a glucose tolerance test (GTT) was performed. HOMA-IR was calculated. Cardiac function and vascular reactivity were evaluated using the Langendorff isolated heart and isolated carotid arteries methods, respectively. 16S rRNA sequencing of stool samples was used to determine gut microbial composition and abundance. HFFD-fed Watanabe rabbits exhibited increased fasting insulin (p < 0.03, 12th week vs. Baseline), HOMA-IR (p < 0.03 vs. Control), area under the curve of the GTT (p < 0.02 vs. Control), triglycerides (p < 0.05, 12th week vs. Baseline), TC (p < 0.01 vs. Control), LDL-C (p < 0.001 vs. Control). The HFFD group also displayed a significant decrease in intestinal microbial richness, evenness and diversity (FDR < 0.001, FDR < 0.0001, FDR < 0.01, respectively vs. Control group) and an increase in its Firmicutes/Bacteroidetes ratio (R = 3.39 in control vs. R = 28.24 in the HFFD group) indicating a shift in intestinal microbial composition and diversity. Our results suggest that HFFD induces insulin resistance and gut microbiota dysbiosis and accentuates dyslipidemia; and that, when subjected to HFFD, Watanabe rabbits might become a potential diet-induced MetS animal models with two main features, dyslipidemia and insulin resistance.

Rabbit models of intracranial atherosclerotic disease for pathological validation of vessel wall MRI.

Vessel wall magnetic resonance imaging can improve the evaluation of intracranial atherosclerotic disease. However, pathological validation is needed to improve vessel wall magnetic resonance imaging techniques. Human pathology samples are not practical for such analysis, so an animal model is therefore needed. Watanabe heritable hyperlipidemic rabbits and apolipoprotein E knockout rabbits were evaluated against New Zealand white wild-type rabbits. Evaluation of intracranial arteries was performed with vessel wall magnetic resonance imaging and pathological analysis, rating the presence and severity of disease in each segment. Two-tailed t-tests were performed to compare disease occurrence and severity prevalence among rabbit subtypes. Sensitivity and specificity were calculated to assess the diagnostic accuracy of vessel wall magnetic resonance imaging. Seventeen rabbits (five Watanabe heritable hyperlipidemic, four apolipoprotein E knockout and eight New Zealand white) were analysed for a total of 51 artery segments. Eleven segments (five Watanabe heritable hyperlipidemic and six apolipoprotein E knockout) demonstrated intracranial atherosclerotic disease on pathology. Disease model animals had lesions more frequently than New Zealand white animals (P<0.001). The sensitivity and specificity of vessel wall magnetic resonance imaging for the detection of intracranial atherosclerotic disease were 68.8% and 95.2%, respectively. When excluding mild cases to assess vessel wall magnetic resonance imaging accuracy for detecting moderate to severe intracranial atherosclerotic disease lesions, sensitivity improved to 100% with unchanged specificity. Intracranial atherosclerotic disease can be reliably produced and detected using 3T vessel wall magnetic resonance imaging-compatible Watanabe heritable hyperlipidemic and ApoE rabbit models. Further analysis is needed to characterize better the development and progression of the disease to correlate tissue-validated animal findings with those in human vessel wall magnetic resonance imaging studies.

Effect of the dipeptidyl peptidase-4 inhibitor linagliptin on atherosclerotic lesions in Watanabe heritable hyperlipidemic rabbits: iMap-IVUS and pathological analysis.

Dipeptidyl peptidase-4 (DPP-4) inhibitors have potential as a treatment for atherosclerosis. However, it is unclear whether DPP-4 inhibitors stabilize atherosclerotic plaque or alter the composition of complex plaque. Sixteen Watanabe heritable hyperlipidemic rabbits aged 10-12weeks with atherosclerotic plaque in the brachiocephalic artery detected by iMap™ intravascular ultrasound (IVUS) were divided into a DPP-4 inhibitor group and a control group. Linagliptin was administered to the DPP-4 inhibitor group via nasogastric tube at a dose of 10mg/kg/day for 16weeks, and control rabbits received the same volume of 0.5% hydroxyethylcellulose. After evaluation by IVUS at 16weeks, the brachiocephalic arteries were harvested for pathological examination. IVUS revealed that linagliptin significantly reduced the plaque volume and vessel volume (control group vs. DPP-4 inhibitor group: ∆plaque volume, 1.02 ± 0.96mm3 vs. - 3.59 ± 0.92 mm3, P = 0.004; ∆vessel volume, - 1.22 ± 2.36mm3 vs. - 8.66 ± 2.33 mm3, P = 0.04; %change in plaque volume, 6.90 ± 5.62% vs. - 15.06 ± 3.29%, P = 0.005). With regard to plaque composition, linagliptin significantly reduced the volume of fibrotic, lipidic, and necrotic plaque (control group vs. DPP-4 inhibitor group: ∆fibrotic volume, 0.56 ± 1.27mm3 vs. - 5.57 ± 1.46mm3, P = 0.04; ∆lipidic volume, 0.24 ± 0.24mm3 vs. - 0.42 ± 0.16mm3 P = 0.04; ∆necrotic volume, 0.76 ± 0.54mm3 vs. - 0.84 ± 0.25mm3, P = 0.02). Pathological examination did not show any significant differences in the %smooth muscle cell area or %fibrotic area, but infiltration of macrophages into plaque was reduced by linagliptin treatment (%macrophage area: 12.03% ± 1.51% vs. 7.21 ± 1.65%, P < 0.05). These findings indicate that linagliptin inhibited plaque growth and stabilized plaque in Watanabe heritable hyperlipidemic rabbits.

Identification and visualization of oxidized lipids in atherosclerotic plaques by microscopic imaging mass spectrometry-based metabolomics

Background and aimsDysregulated lipid metabolism has emerged as one of the major risk factors of atherosclerosis. Presently, there is a consensus that oxidized LDL (oxLDL) promotes development of atherosclerosis and downstream chronic inflammatory responses. Due to the dynamic metabolic disposition of lipoprotein, conventional approach to purify bioactive lipids for subsequent comprehensive analysis has proven to be inadequate for elucidation of the oxidized lipids species accountable for pathophysiology of atherosclerotic lesions. Herein, we aimed to utilize a novel mass microscopic imaging technology, coupled with mass spectrometry (MS) to characterize oxidized lipids in atherosclerotic lesions. MethodsWe attempted to use MALDI-TOF-MS and iMScope to identify selected oxidized lipid targets and visualize their respective localizations in study models of atherosclerosis. ResultsBased on the MS analysis, detection of 7-K under positive ionization through product ion peak at m/z 383 [M + H–H2O] indicated the distinctive presence of targeted lipid within Cu2+-oxLDL and Cu2+-oxLDL loaded macrophage-like J774A.1 cells, along with other cholesterol oxidation products. Moreover, the application of two-dimensional iMScope has successfully visualized the localization of lipids in aortic atherosclerotic plaques of the Watanabe heritable hyperlipidemic (WHHL) rabbit. Distinctive lipid distribution profiles were observed in atherosclerotic lesions of different sizes, especially the localizations of lysoPCs in atherosclerotic plaques. ConclusionsTaken together, we believe that both MALDI-TOF-MS and iMScope metabolomics technology may offer a novel proposition for future pathophysiological studies of lipid metabolism in atherosclerosis.

Apolipoprotein E knockout rabbit model of intracranial atherosclerotic disease.

Intracranial atherosclerotic disease (ICAD) is the most common cause of ischemic stroke. Poor understanding of the disease due to limited human data leads to imprecise treatment. Apolipoprotein E knockout (ApoE‐KO) rabbits were compared to an existing model, the Watanabe heritable hyperlipidemic (WHHL) rabbit, and wild‐type New Zealand white (NZW) rabbit controls. Intracranial artery samples were assessed on histopathology for the presence of ICAD. Logistic and ordinal regression analyses were performed to assess for disease presence and severity, respectively. Eighteen rabbits and 54 artery segments were analyzed. Univariate logistic analysis confirmed the presence of ICAD in model rabbits (P < .001), while no difference was found between WHHL and ApoE‐KO rabbits (P = .178). In multivariate analysis, only classification as a model vs wild‐type animal (P < .001) was associated with the presence of ICAD. Univariate ordinal regression analysis demonstrated an association between ICAD severity and model animals (P = .001), with no difference was noted between WHHL and ApoE‐KO rabbits (P = .528). In multivariate ordinal regression analysis, only classification as a model retained significance (P < .001). ICAD can be reliably produced in ApoE‐KO rabbits, developing the disease comparably to the older WHHL model. Further analysis is warranted to optimize accelerated development of ICAD in ApoE‐KO rabbits to more efficiently study this disease.

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.

The History of the WHHL Rabbit, an Animal Model of Familial Hypercholesterolemia (I) - Contribution to the Elucidation of the Pathophysiology of Human Hypercholesterolemia and Coronary Heart Disease -

Animal models that closely resemble both human disease findings and their onset mechanism have contributed to the advancement of biomedical science. The Watanabe heritable hyperlipidemic (WHHL) rabbit and its advanced strains (the coronary atherosclerosis-prone and the myocardial infarction-prone WHHL rabbits) developed at Kobe University (Kobe, Japan), an animal model of human familial hypercholesterolemia, have greatly contributed to the elucidation of the pathophysiology of human lipoprotein metabolism, hypercholesterolemia, atherosclerosis, and coronary heart disease, as described below. 1) The main part of human lipoprotein metabolism has been elucidated, and the low-density lipoprotein (LDL) receptor pathway hypothesis derived from studies using fibroblasts was proven in vivo. 2) Oxidized LDL accumulates in the arterial wall, monocyte adhesion molecules are expressed on arterial endothelial cells, and monocyte-derived macrophages infiltrate the arterial intima, resulting in the formation and progression of atherosclerosis. 3) Coronary lesions differ from aortic lesions in lesion composition. 4) Factors involved in the development of atherosclerosis differ between the coronary arteries and aorta. 5) The rupture of coronary lesions requires secondary mechanical forces, such as spasm, in addition to vulnerable plaques. 6) Specific lipid molecules in the blood have been identified as markers of the progression of coronary lesions. At the end of the breeding of the WHHL rabbit family at Kobe University, this review summarizes the history of the development of the WHHL rabbit family and their contribution to biomedical science.

Bile-duct proliferation as an unexpected side-effect after AAV2-LDLR gene transfer to rabbit liver

Familial hypercholesterolemia (FH) is an inherited disease of lipoprotein metabolism caused by a defect in the LDL receptor (LDLR) leading to severe hypercholesterolemia, and associated with an increased risk of coronary heart disease and myocardial infarction. We have developed a gene therapy protocol for FH using AAV2, AAV9 and lentiviral vectors and tested safety and efficacy in LDL receptor deficient Watanabe Heritable Hyperlipidemic rabbits. We show that LV-LDLR produced a significant long-lasting decrease in total serum cholesterol whereas AAV9-LDLR resulted only in a transient decrease and AAV2-LDLR failed to reduce serum cholesterol levels. A significant pathological side effect, bile-duct proliferation, was seen in the liver of AAV2-LDLR rabbits associated with an increased expression of Cyr61 matricellular protein. Special attention should be given to liver changes in gene therapy applications when genes affecting cholesterol and lipoprotein metabolism are used for therapy.

Integral role of receptor for advanced glycation end products (RAGE) in nondiabetic atherosclerosis.

An advanced glycation end products (AGE)/a receptor for AGE (RAGE) axis plays a central role in the pathogenesis of diabetic vascular remodeling. This study was conducted to clarify the role of RAGE in nondiabetic atherosclerosis. We used the aortic and coronary atherosclerotic lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits prone to myocardial infarction (WHHLMI) at 1 to 14 months. Immunohistochemistry demonstrated the significant expression of RAGE as early as at 1 month with the stronger expression at 3 and 7 months, which was remarkably diminished at 14 months. RAGE expression was concordant with AGE accumulation. The major original sources of RAGE expression were macrophages and smooth muscle cells in addition to endothelial cells, and RAGE expression was distributed in the areas of phospholipid products, a component of oxidized LDL and nitrotyrosine. The concentrations of serum AGE did not alter significantly with aging. These findings suggested the expression of RAGE was induced by hyperlipidemia and oxidative stress independent of diabetes in WHHLMI rabbits. Additionally, our in vitro study showed that silencing of RAGE tended to attenuate oxidized-LDL-triggered PAI-1 expression in human cultured macrophages, as well as oxidized-LDL-induced tissue factor expression in peritoneal macrophages, suggesting a possible role of RAGE in prothrombogenic molecular regulation. In conclusion, the present study provides in vivo evidence that RAGE plays an integral role in the initiation and progression of nondiabetic atherosclerosis, suggesting that RAGE may be a novel target for treating not only diabetic but also nondiabetic vascular complications.

Genomic and Transcriptomic Analysis of Hypercholesterolemic Rabbits: Progress and Perspectives.

Rabbits (Oryctolagus cuniculus) are one of the most widely used animal models for the study of human lipid metabolism and atherosclerosis because they are more sensitive to a cholesterol diet than other experimental animals such as rodents. Currently, two hypercholesterolemic rabbit models are frequently used for atherosclerosis studies. One is a cholesterol-fed wild-type rabbit and the other is the Watanabe heritable hyperlipidemic (WHHL) rabbit, which is genetically deficient in low density lipoprotein (LDL) receptor function. Wild-type rabbits can be easily induced to develop severe hypercholesterolemia with a cholesterol-rich diet due to the marked increase in hepatically and intestinally derived remnant lipoproteins, called β-very low density lipoproteins (VLDL), which are rich in cholesteryl esters. WHHL rabbits are characterized by elevated plasma LDL levels on a standard chow diet, which resembles human familial hypercholesterolemia. Therefore, both rabbit models develop aortic and coronary atherosclerosis, but the elevated plasma cholesterol levels are caused by completely different mechanisms. In addition, cholesterol-fed rabbits but not WHHL rabbits exhibit different degrees of hepatosteatosis. Recently, we along with others have shown that there are many differentially expressed genes in the atherosclerotic lesions and livers of cholesterol-fed rabbits that are either significantly up- or down-regulated, compared with those in normal rabbits, including genes involved in the regulation of inflammation and lipid metabolism. Therefore, dietary cholesterol plays an important role not only in hypercholesterolemia and atherosclerosis but also in hepatosteatosis. In this review, we make an overview of the recent progress in genomic and transcriptomic analyses of hypercholesterolemic rabbits. These transcriptomic profiling data should provide novel insight into the relationship between hypercholesterolemia and atherosclerosis or hepatic dysfunction caused by dietary cholesterol.

Hypertension Enhances Advanced Atherosclerosis and Induces Cardiac Death in Watanabe Heritable Hyperlipidemic Rabbits

Hypertension is a major risk factor for the development of atherosclerosis. Cardiovascular risk has been reported to be significantly increased in hyperlipidemic patients with hypertension. However, it is not clear whether hypertension can directly destabilize plaques, thereby enhancing cardiovascular events. To examine whether hypertension enhances the development of atherosclerosis and increases plaque vulnerability, we generated hypertensive Watanabe heritable hyperlipidemic (WHHL) rabbits by surgical removal of one kidney and partial ligation of the other renal artery and compared the nature of aortic and coronary atherosclerosis in hypertensive WHHL rabbits with normotensive WHHL rabbits. All hypertensive WHHL rabbits died from 34 to 56 weeks after surgery, whereas no normotensive WHHL rabbits died. Pathologic examinations revealed that hypertensive WHHL rabbits showed different degrees of myocardial infarction caused by severe coronary stenosis along with myocardial hypertrophy. Furthermore, aortic lesions in hypertensive WHHL rabbits exhibited a higher frequency of intraplaque hemorrhage and vulnerable plaques than those in normotensive WHHL rabbits. These results indicate that hypertension induced by the surgical removal of one kidney and partial ligation of the other renal artery method in WHHL rabbits may not only enhance the development of atherosclerosis but also destabilize the plaques, increasing cardiac death.

Long-term atorvastatin treatment decreases heart maximal oxygen consumption and its vulnerability to in vitro oxidative stress in Watanabe heritable hyperlipidemic rabbit.

Statins are currently used in prevention of cardiovascular diseases in high-risk populations, and could be considered in primary prevention. However, few studies are available on the long-term effects of low doses of statins, especially on mitochondrial function and reactive oxygen species (ROS) metabolism at cardiac level. This study aimed to determine potential effects of a long-term atorvastatin treatment, at low-dose concentration, on the myocardium mitochondrial respiration. Thirty-four Watanabe rabbits were treated or not with atorvastatin (2.5 mg·kg-1·day-1) from the age of 3 to 12 months. Every 3 months, proton leak, basal (V0), and maximal (Vmax) mitochondrial respiration on cardiac permeabilized fibers were measured. Additionally, the vulnerability to ROS, cardiac enzymatic antioxidant defenses, and oxidative damage (lipoperoxidation) were analyzed. Proton leak increased over the duration of the experiment (up to 60% from Vmax at 12 months). Moreover, the statin treatment induced a decrease of Vmax and a decrease of ROS susceptibility of cardiac mitochondria. However, the lipoperoxidation and the antioxidant defenses were not dependent on the presence of statin treatment, or on its duration. This is the first study showing a protective effect of long-term statins treatment against the ROS susceptibility in the cardiac muscle.

Abstract 622: Expression of Fatty Acid Binding Protein-4 in Atherosclerotic Watanabe Heritable Hyperlipidemic (WHHL) Rabbit Aortas

Background: Atherosclerotic diseases are among the prime cause of mortality globally. Proper animal model that can resemble the lipid metabolism and atherosclerotic lesions in humans is key to study the pathophysiology of the disease. One of the most famous of these atherosclerotic animal models is Watanabe heritable hyperlipidemic (WHHL) rabbits. They are deficient of LDL receptors, hence develop spontaneous hypocholesterolemia and atherosclerosis. Previous studies have demonstrated the importance of Fatty acid binding protein-4 (FABP4), a lipid chaperon that plays an important role in atherosclerotic diseases. However, FABP4 expression or role in WHHL rabbits was not investigated yet. Hence, we hypothesis that WHHL rabbits express FABP4 in their aortas and targeting FABP4 acts as a novel target to treat atherosclerosis. Materials and Methods: Two WHHL rabbits of greater than one year old were sacrificed and aortas were isolated. One of the aorta was flash frozen and other was formalin fixed. Proteins were extracted from frozen section. Western blot was performed to estimate the concentration of FABP4. Immunohistochemistry was performed on the formalin fixed tissue to analyze the expression of FABP4. Results: WHHL aortas having noteworthy amount of atherosclerotic plaques expressed FABP4 supporting the importance of FABP4 in cardiovascular diseases. Conclusion: WHHL rabbits which are model of atherosclerosis expresses FABP4 in their aortas thus paving the way to use this model to target FABP4 for treating atherosclerosis.

Progression of calcific aortic valve sclerosis in WHHLMI rabbits

Background and aimsAortic valve stenosis (AS) is the most common valvular heart disease and can be life-threatening. The pathogenesis of aortic valve calcification remains largely unknown, primarily due to the lack of an adequate animal model. The high-cholesterol diet-induced AS model in rabbits is one of the established models, but it has the significant limitation of liver dysfunction leading to low survival rates. We hypothesized that a myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbit, an animal model of familial hypercholesterolemia and atherosclerosis, is a useful animal model of AS. MethodsWHHLMI rabbits, aged 20 months and 30 months (n = 19), and control Japanese White rabbits (n = 4), aged 30 months, were used and evaluated by echocardiography under anesthesia. Pathological evaluation and quantitative analyses by polymerase chain reaction (PCR) were also performed. ResultsThe lipid profile was similar between 20 months and 30 months. Two rabbits died due to spontaneous myocardial infarction during the study. Thirty-month-old WHHLMI rabbits exhibited significantly smaller aortic valve area (0.22 ± 0.006 cm2vs. 0.12 ± 0.01 cm2, p < 0.05) and higher maximal transvalvular pressure gradient (7.0 ± 0.32 vs. 9.9 ± 0.95 mmHg, p < 0.05) than 20 month-old rabbits. Macroscopic examination of excised aortic valves demonstrated thickened and degenerated valve leaflets at 30 months. Histological evaluation confirmed thickened leaflets with calcified nodules at 30 months. Real-time PCR of resected aortic valve also showed increased expression level of calcification-related molecules including osteopontin, Sox9, Bmp2, RANKL, osteoprotegerin, and Runx2 (p < 0.05 each) in 30-month-old rabbits. ConclusionsWHHLMI rabbits may be useful models of early-stage AS in vivo.

Transplantation of genetically modified hepatocytes after liver preconditioning in Watanabe heritable hyperlipidemic rabbit

BackgroundHepatocyte transplantation is a potentially less invasive alternative to liver transplantation for treating inherited metabolic liver diseases. We developed an autotransplantation protocol of ex vivo genetically modified hepatocytes combining lentiviral transduction and transplantation after liver preconditioning by partial portal vein embolization. We investigated the metabolic efficiency of this approach in Watanabe rabbits, animal model of familial hypercholesterolemia. MethodsOur autotransplantation experimental protocol was used in two groups of rabbits (n = 10), experimental and sham, receiving transduced and control hepatocytes, respectively. Isolated hepatocytes from left liver lobes were transduced using recombinant lentiviruses. Median lobe portal branches were embolized under fluoroscopic control. Functional measurement of low-density lipoprotein (LDL) receptor expression was assessed by LDL internalization assays. Cholesterol level evolution was monitored. Rabbits were killed 20 wk after the procedure. ResultsThree rabbits of each group died several hours after hepatocyte transplantation; autopsy revealed portal vein thrombosis in two rabbits from each group. The protocol was therefore modified with hepatocytes being transplanted through splenic injection. Lentiviral hepatocyte transduction efficacy was 64.5%. Fluorescence microscopy revealed Dil-LDL internalization of transduced hepatocytes. Seven rabbits in each group were considered for lipid analysis. Four weeks after autotransplantation, median total cholesterol level decreased in the experimental group, without reaching statistical significance (8.9 [8.0-9.8] g/L versus 6.3 [0.5-8.3]; P = 0.171). In the experimental group, enzyme-linked immunosorbent assay detected significant antibody expression against human low-density lipoprotein receptor. ConclusionsAutotransplantation protocol allowed a nonstatistically significant improvement of the lipid profile in Watanabe rabbits. Further experiments involving a larger number of animals are necessary to confirm or refute our findings.

Inhibition of plaque progression and promotion of plaque stability by glucagon-like peptide-1 receptor agonist: Serial in vivo findings from iMap-IVUS in Watanabe heritable hyperlipidemic rabbits

Background and aimsGlucagon-like peptide-1 (GLP-1) is thought to inhibit development of aortic atherosclerosis and plaque formation. However, whether GLP-1 stabilizes fully developed atherosclerotic plaque or alters the complicated plaque composition remains unclarified. MethodsTen Watanabe heritable hyperlipidemic (WHHL) rabbits were divided into GLP-1 receptor agonist treatment group and control group. After confirmation of atherosclerotic plaques in brachiocephalic arteries by iMap intravascular ultrasound (iMAP-IVUS), GLP-1 receptor agonist lixisenatide was administered to WHHL rabbits at 30 nmoL/kg/day for 12 weeks by osmotic pump. An equal volume of normal saline was administered in a control group. After evaluation by iMAP-IVUS at 12 weeks, brachiocephalic arteries were harvested for pathological histological analysis. ResultsiMAP-IVUS analysis revealed larger fibrotic plaque components and smaller necrotic and calcified plaque components in the GLP-1 group than in the control group; %fibrotic area: 66.30 ± 2.06% vs. 75.14 ± 2.62%, p < 0.01, %necrotic area: 23.25 ± 1.87% vs. 16.17 ± 2.27%, p = 0.02, %calcified area: 2.15 ± 0.24% vs. 1.00 ± 0.18%, p < 0.01), indicating that GLP-1 receptor agonist might modify plaque composition and increase plaque stability. Histological analysis confirmed that GLP-1 receptor agonist treatment improved smooth muscle cell (SMC)-rich plaque with increased fibrotic content. Furthermore, plaque macrophage infiltration and calcification were significantly reduced by GLP-1 receptor agonist treatment; %SMC area: 6.93 ± 0.31% vs. 8.14 ± 0.48%, p = 0.02; %macrophage area: 9.11 ± 0.80% vs. 6.19 ± 0.85%, p < 0.01; %fibrotic area: 54.75 ± 1.63% vs. 69.60 ± 2.12%, p = 0.02; %calcified area: 3.25 ± 0.67% vs. 0.75 ± 0.15%, p = 0.02). ConclusionsGLP-1 receptor agonist inhibited plaque progression and promoted plaque stabilization by inhibiting plaque growth and modifying plaque composition.