Manabu Niimi

Human Apolipoprotein A-II Protects Against Diet-Induced Atherosclerosis in Transgenic Rabbits

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

ApoE knockout rabbits: A novel model for the study of human hyperlipidemia

OBJECTIVE Rabbits are one of the best animal models for the study of hyperlipidemia and atherosclerosis. Although many transgenic rabbits have been created, the development of gene knockout (KO) rabbits has been impossible due to the lack of rabbit embryonic stem cells. We along with others recently generated KO rabbits using genome editing techniques. In the current study, we characterized the lipoprotein profiles of apoE KO rabbits on both chow and cholesterol diets and investigated their susceptibility to a diet-induced atherosclerosis. APPROACH AND RESULTS We analyzed plasma lipids and lipoproteins of apoE KO rabbits and compared them with those of wild-type rabbits. On a chow diet, homozygous (but not heterozygous) apoE KO rabbits showed mild hyperlipidemia and, when challenged with a cholesterol diet, they showed greater susceptibility to diet-induced hyperlipidemia than did the wild-type rabbits and their plasma total cholesterol levels were remarkably increased (1070 ± 61 mg/dL in apoE KO vs. 169 ± 79 mg/dL in the wild type, p < 0.001). Hyperlipidemia in apoE KO rabbits was caused by elevated remnant lipoproteins. Interestingly, increased remnant lipoproteins in apoE KO rabbits were predominated by apoB-48 and rich in both apoA-I and apoA-IV contents. Furthermore, apoE KO rabbits developed greater aortic atherosclerosis than wild-type rabbits when fed with a cholesterol diet for 10 weeks. CONCLUSIONS To our knowledge, this is the first report of generating KO rabbits for the study of lipid and lipoprotein metabolism. ApoE KO rabbits should be a useful model for the study of human hyperlipidemia and atherosclerosis.

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.

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. Copyright

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.

Deficiency of Cholesteryl Ester Transfer Protein Protects Against Atherosclerosis in Rabbits

Objective— CETP (cholesteryl ester transfer protein) plays an important role in lipoprotein metabolism; however, whether inhibition of CETP activity can prevent cardiovascular disease remains controversial. Approach and Results— We generated CETP knockout (KO) rabbits by zinc finger nuclease gene editing and compared their susceptibility to cholesterol diet–induced atherosclerosis to that of wild-type (WT) rabbits. On a chow diet, KO rabbits showed higher plasma levels of high-density lipoprotein (HDL) cholesterol than WT controls, and HDL particles of KO rabbits were essentially rich in apolipoprotein AI and apolipoprotein E contents. When challenged with a cholesterol-rich diet for 18 weeks, KO rabbits not only had higher HDL cholesterol levels but also lower total cholesterol levels than WT rabbits. Analysis of plasma lipoproteins revealed that reduced plasma total cholesterol in KO rabbits was attributable to decreased apolipoprotein B–containing particles, while HDLs remained higher than that in WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. Apolipoprotein B–depleted plasma isolated from CETP KO rabbits showed significantly higher capacity for cholesterol efflux from macrophages than that from WT rabbits. Furthermore, HDLs isolated from CETP KO rabbits suppressed tumor necrosis factor-&agr;–induced vascular cell adhesion molecule 1 and E-selectin expression in cultured endothelial cells. Conclusions— These results provide evidence that genetic ablation of CETP activity protects against cholesterol diet–induced atherosclerosis in rabbits.

Angiotensin II Destabilizes Coronary Plaques in Watanabe Heritable Hyperlipidemic Rabbits

Objective—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. Approach and Results—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. Conclusions—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.

Increased Hepatic Expression of Endothelial Lipase Inhibits Cholesterol Diet–Induced Hypercholesterolemia and Atherosclerosis in Transgenic Rabbits

Objective— Endothelial lipase (EL) is a key determinant in plasma high-density lipoprotein-cholesterol. However, functional roles of EL on the development of atherosclerosis have not been clarified. We investigated whether hepatic expression of EL affects plasma lipoprotein metabolism and cholesterol diet–induced atherosclerosis. Approach and Results— We generated transgenic (Tg) rabbits expressing the human EL gene in the liver and then examined the effects of EL expression on plasma lipids and lipoproteins and compared the susceptibility of Tg rabbits with cholesterol diet–induced atherosclerosis with non-Tg littermates. On a chow diet, hepatic expression of human EL in Tg rabbits led to remarkable reductions in plasma levels of total cholesterol, phospholipids, and high-density lipoprotein-cholesterol compared with non-Tg controls. On a cholesterol-rich diet for 16 weeks, Tg rabbits exhibited significantly lower hypercholesterolemia and less atherosclerosis than non-Tg littermates. In Tg rabbits, gross lesion area of aortic atherosclerosis was reduced by 52%, and the lesions were characterized by fewer macrophages and smooth muscle cells compared with non-Tg littermates. Conclusions— Increased hepatic expression of EL attenuates cholesterol diet–induced hypercholesterolemia and protects against atherosclerosis.

Glutathione inhibits antibody and complement-mediated immunologic cell injury via multiple mechanisms

Antioxidant glutathione (GSH) plays an important role in the regulation of immunity. However, little is known about its effects on humoral immunity, especially its action on effector molecules like antibody and complement. Given that these molecules contain abundant disulfide bonds, we speculated that GSH might influence the action of these proteins via its thiol function. Using a model of a glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we addressed this hypothesis. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused a complement-dependent cell lysis, which was completely blocked by GSH. Moreover, GSH potently prevented the antibody-mediated agglutination of red blood cells and aggregation of antibody-sensitized microspheres. Further analysis revealed that GSH inhibited antibody binding to antigens and promoted the conversion of the antibodies to its reduced forms. GSH also potently inhibited the formation and deposition of C5b-9 in MCs and suppressed both the classic and alternative complement activation pathway. Lastly, GSH attenuated P38 activation, an oxidative sensitive kinase that partially mediated the antibody- and complement-dependent MC lysis. Depletion of GSH via inhibiting gamma-glutamylcysteine synthetase or xCT transporter augmented P38 activation and sensitized MCs to the cell lysis. Collectively, our results indicate that GSH protects cells from immunological cell damage via mechanisms involving inhibition of antibody binding to the antigens, suppression of complement activation and augmentation of cellular defense mechanism. Our study provides novel mechanistic insights into the actions of GSH in the regulation of immune responses and suggests that GSH might be used to treat certain immune disorders.

Principles and Applications of Rabbit Models for Atherosclerosis Research

Rabbits are one of the most used experimental animals for biomedical research, particularly as a bioreactor for the production of antibodies. However, many unique features of the rabbit have also made it as an excellent species for examining a number of aspects of human diseases such as atherosclerosis. Rabbits are phylogenetically closer to humans than rodents, in addition to their relatively proper size, tame disposition, and ease of use and maintenance in the laboratory facility. Due to their short life spans, short gestation periods, high numbers of progeny, low cost (compared with other large animals) and availability of genomics and proteomics, rabbits usually serve to bridge the gap between smaller rodents (mice and rats) and larger animals, such as dogs, pigs and monkeys, and play an important role in many translational research activities such as pre-clinical testing of drugs and diagnostic methods for patients. The principle of using rabbits rather than other animals as an experimental model is very simple: rabbits should be used for research, such as translational research, that is difficult to accomplish with other species. Recently, rabbit genome sequencing and transcriptomic profiling of atherosclerosis have been successfully completed, which has paved a new way for researchers to use this model in the future. In this review, we provide an overview of the recent progress using rabbits with specific reference to their usefulness for studying human atherosclerosis.