Watanabe Heritable Hyperlipidemic 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.

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.

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.

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.

In Vivo Translation of the CIRPI System: Revealing Molecular Pathology of Rabbit Aortic Atherosclerotic Plaques.

Thin-cap fibroatheroma (TCFA) are the unstable lesions in coronary artery disease that are prone to rupture, resulting in substantial morbidity and mortality worldwide. However, their small size and complex morphologic and biologic features make early detection and risk assessment difficult. We tested our newly developed catheter-based Circumferential-Intravascular-Radioluminescence-Photoacoustic-Imaging (CIRPI) system in vivo to enable detection and characterization of vulnerable plaque structure and biology in rabbit abdominal aorta. Methods: The CIRPI system includes a novel optical probe combining circumferential radioluminescence imaging and photoacoustic tomography (PAT). The probe's CaF2:Eu-based scintillating imaging window captures radioluminescence images (360° view) of plaques by detecting β-particles during 18F-FDG decay. A tunable laser-based PAT characterizes tissue constituents of plaque at 7 different wavelengths-540 and 560 nm (calcification), 920 nm (cholesteryl ester), 1040 nm (phospholipids), 1180 nm (elastin/collagen), 1210 nm (cholesterol), and 1235 nm (triglyceride). A single B-scan is concatenated from 330 A-lines captured during a 360° rotation. The abdominal aorta was imaged in vivo in both atherosclerotic rabbits (Watanabe Heritable Hyper Lipidemic [WHHL], 13-mo-old male, n = 5) and controls (New Zealand White, n = 2). Rabbits were fasted for 6 h before 5.55 × 107 Bq (1.5 mCi) of 18F-FDG were injected 1 h before the imaging procedure. Rabbits were anesthetized, and the right or left common carotid artery was surgically exposed. An 8 French catheter sheath was inserted into the common carotid artery, and a 0.035-cm (0.014-in) guidewire was advanced to the iliac artery, guided by x-ray fluoroscopy. A bare metal stent was implanted in the dorsal abdominal aorta as a landmark, followed by the 7 French imaging catheters that were advanced up to the proximal stent edge. Our CIRPI and clinical optical coherence tomography (OCT) were performed using pullback and nonocclusive flushing techniques. After imaging with the CIRPI system, the descending aorta was flushed with contrast agent, and OCT images were obtained with a pullback speed of 20 mm/s, providing images at 100 frames/s. Results were verified with histochemical analysis. Results: Our CIRPI system successfully detected the locations and characterized both stable and vulnerable aortic plaques in vivo among all WHHL rabbits. Calcification was detected from the stable plaque (540 and 560 nm), whereas TCFA exhibited phospholipids/cholesterol (1040 nm, 1210 nm). These findings were further verified with the clinical OCT system showing an area of low attenuation filled with lipids within TCFA. PAT images illustrated broken elastic fiber/collagen that could be verified with the histochemical analysis. All WHHL rabbits exhibited sparse to severe macrophages. Only 4 rabbits showed both moderate-to-severe level of calcifications and cholesterol clefts. However, all rabbits exhibited broken elastic fibers and collagen deposition. Control rabbits showed normal wall thickness with no presence of plaque tissue compositions. These findings were verified with OCT and histochemical analysis. Conclusion: Our novel multimodality hybrid system has been successfully translated to in vivo evaluation of atherosclerotic plaque structure and biology in a preclinical rabbit model. This system proposed a paradigm shift that unites molecular and pathologic imaging technologies. Therefore, the system may enhance the clinical evaluation of TCFA, as well as expand our understanding of coronary artery disease.

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.

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.

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.

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.

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.

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.