Enqi Liu

Macrophage Metalloelastase Accelerates the Progression of Atherosclerosis in Transgenic Rabbits

Background— Macrophage metalloelastase (matrix metalloproteinase [MMP]-12) is upregulated in atherosclerotic lesions and aneurysm; thus, increased MMP-12 activity may play an important role in the pathogenesis of atherosclerosis. However, the pathological roles of MMP-12 in the initiation and progression of atherosclerosis have not been defined. Methods and Results— We compared the susceptibility of MMP-12 transgenic (Tg) rabbits to cholesterol-rich diet–induced atherosclerosis with that of non-Tg littermate rabbits. The rabbits were maintained at either relatively lower levels of hypercholesterolemia for shorter periods or higher levels of hypercholesterolemia for longer periods through a diet containing different amounts of cholesterol. We found no significant difference in the aortic atherosclerotic lesion size or quality between Tg and non-Tg rabbits at lower hypercholesterolemia. At higher hypercholesterolemia for longer periods, however, Tg rabbits developed more extensive atherosclerosis in the aortas and coronary arteries than did non-Tg rabbits. Histological examinations revealed that atherosclerotic lesions of Tg rabbits contained prominent macrophage infiltration associated with marked disruption of the elastic lamina in the tunica media with occasional formation of aneurysm-like lesions. Furthermore, increased expression of MMP-12 derived from macrophages was associated with elevated expression of MMP-3, suggesting that MMP-12 may play a pivotal role in the cascade activation of other MMPs, thereby exacerbating extracellular matrix degradation during the progression of atherosclerosis. Conclusions— Overexpression of MMP-12 causes accelerated atherosclerosis in Tg rabbits. These results suggest that macrophage-derived MMP-12 participates in the progression of atherosclerosis.

Increased Expression of Vascular Endothelial Growth Factor in Kidney Leads to Progressive Impairment of Glomerular Functions

Vascular endothelial growth factor (VEGF) is an important mediator in maintaining normal kidney functions. In addition, several lines of evidence suggest that upregulation of VEGF in glomeruli may be associated with or cause renal dysfunction such as diabetic nephropathy. For elucidation of the pathologic consequences of high levels of VEGF in glomeruli, transgenic (Tg) rabbits that express human VEGF(165) isoform in both kidney and liver under the control of the human alpha-1-antitrypsin promoter were generated and characterized. With the use of heterozygous Tg rabbits and their littermates aged 8 to 55 wk, renal functions and structures were investigated. Compared with control rabbits, Tg rabbits exhibited progressive proteinuria with increased GFR at the early stage and decreased GFR at the later stage. Histologic examinations revealed that Tg rabbit kidneys were characterized by considerable glomerular hypertrophy as a result of increased proliferation of both glomerular capillaries and mesangial cells accompanied by prominent podocyte hypertrophy. With increasing age starting from 20 wk, Tg rabbit kidneys showed prominent formation of microaneurysms and capillary proliferation at the vascular pole area. At a later stage (55 wk), many glomeruli showed sclerosis and tuft collapse with the formation of glomerular cysts on a background of tubular atrophy and interstitial fibrosis. This study provides the first evidence that increased expression of VEGF in glomeruli directly causes the glomerular hypertrophy that is associated with proteinuria, suggesting that VEGF exerts multiple effects on the glomerular pathophysiologic processes.

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

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

Diet-induced central obesity and insulin resistance in rabbits.

The present study was designed to examine whether rabbits fed a diet containing high fat and sucrose could develop obesity and insulin resistance (IR), the major pathophysiological features of metabolic syndrome. Male Japanese white rabbits were fed either a normal chow diet (control) or high fat and sucrose diet (HFSD) for 36 weeks. Plasma levels of triglycerides (TG), total cholesterol (TC), glucose and insulin were measured. To evaluate glucose metabolism, we performed an intravenous glucose tolerance test. In addition, we compared adipose tissue accumulation in HFSD-fed rabbits with that in normal rabbits. HFSD constantly and significantly led to an increase in body weight of HFSD-fed rabbits, caused by significantly higher visceral adipose tissue accumulation. Although there were no differences in plasma TG, TC, glucose, insulin levels and blood pressure between the two groups, HFSD-fed rabbits showed impaired glucose clearance associated with higher levels of insulin secretion compared to control rabbits. Our results showed that HFSD induced IR and increased adipose accumulation in rabbits, suggesting that HFSD-fed rabbits may become a model for research on human IR and obesity.

High-fat diet without excess calories induces metabolic disorders and enhances atherosclerosis in rabbits.

OBJECTIVE Excess intake of a high-fat diet (HFD) is associated with obesity and metabolic syndrome, which are major risk factors for cardiovascular disease. However, it is unclear whether consumption of an HFD at a normal calorific range would be detrimental to metabolism or affect the development of atherosclerosis. Here, we tested the hypothesis that consumption of a normal-calorie HFD would impair lipid metabolism, insulin sensitivity, and blood pressure. METHODS AND RESULTS Rabbits fed with an HFD containing either 3% (15% kcal from fat) or 10% (25.8% kcal from fat) coconut oil were compared with control rabbits fed with a standard chow diet (9.3% kcal from fat). All rabbits consumed an equal amount of calories of their respective food. However, HFD feeding induced marked metabolic disorders including increased plasma levels of free fatty acids, insulin resistance, and hypertension compared with control rabbits. Metabolic disorders were more pronounced in 10%-HFD-fed rabbits than 3%-HFD-fed rabbits. To examine whether these disorders affected the development of atherosclerosis, two HFD groups were further fed with a diet containing 0.3% cholesterol for another 18 weeks. We found that 10%-HFD group showed a prominent accumulation of adipose tissue and developed 2-fold greater aortic atherosclerosis than 3%-HFD group. CONCLUSIONS These results suggest that consuming an HFD containing even a normal number of calories can cause insulin resistance, hypertension, and adipose accumulation even without obesity. High amounts of fat in diets apparently accelerate the development of atherosclerosis.

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.

Expression Systems and Species Used for Transgenic Animal Bioreactors

Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm cocoon), the mammary glands of transgenic animals have enormous potential. Compared with other mammalian species (pig, goat, sheep, and cow) that are currently being studied as bioreactors, rabbits offer many advantages: high fertility, easy generation of transgenic founders and offspring, insensitivity to prion diseases, relatively high milk production, and no transmission of severe diseases to humans. Noticeably, for a small- or medium-sized facility, the rabbit system is ideal to produce up to 50 kg of protein per year, considering both economical and hygienic aspects; rabbits are attractive candidates for the mammary-gland-specific expression of recombinant proteins. We also reviewed recombinant proteins that have been produced by targeted expression in the mammary glands of rabbits and discussed the limitations of transgenic animal bioreactors.

Chemopreventive effect of saikosaponin-d on diethylinitrosamine-induced hepatocarcinogenesis: Involvement of CCAAT/enhancer binding protein β and cyclooxygenase-2

Cyclooxygenase-2 (COX-2) and CCAAT/enhancer binding protein β (C/EBPβ) have been shown to be involved in inflammation and carcinogenesis, and our previous study revealed that they were co-overexpressed in human hepatocellular carcinoma (HCC) tissue and a positive correlation was found. Saikosaponin-d (SSD), a triterpene saponin extracted from Bupleurum falcatum L. (Umbelliferae), is known to exert inhibitory effects on COX-2 expression, together with inflammation and hepatic fibrosis. These findings prompted us to investigate the chemopreventive potential of SSD against hepatocarcinogenesis and its possible molecular mechanism in vivo. An experimental model with diethylinitrosamine (DEN)-treated Sprague Dawley rats was used in the present study. DEN (50 mg/kg body weight) and SSD (2 mg/kg body weight) were intraperitoneally injected weekly and daily, respectively. Administration of SSD alone had no side effects. The liver nodule formation, tumorous invasion to surrounding organs and increased cellular atypia induced by DEN were markedly reduced by SSD in the SSD + DEN group compared with the DEN group. On the other hand, immunohistochemical staining demonstrated that the expression of COX-2 and C/EBPβ proteins was significantly increased in tumor cells and macrophages of liver tissue from DEN-treated rats, whereas the expression of the two proteins was markedly lowered in the SSD + DEN group. Overall, our results suggest that SSD prevents DEN-induced hepatocarcinogenesis in rats through inhibition of C/EBPβ and COX-2, providing indispensable experimental evidence for the clinical application of SSD as a novel chemopreventive agent against HCC in the future.

Expression of Human ApoAII in Transgenic Rabbits Leads to Dyslipidemia. A New Model for Combined Hyperlipidemia

Objective—Apolipoprotein AII (apoAII) is the second major apolipoprotein in high-density lipoprotein (HDL). However, the physiological functions of apoAII in lipoprotein metabolism have not been fully elucidated. Methods and Results—We generated human apoAII transgenic (Tg) rabbits, a species that normally does not have an endogenous apoAII gene. Plasma levels of human apoAII in Tg rabbits were ≈30 mg/dL, similar to the plasma levels in healthy humans. The expression of human apoAII in Tg rabbits resulted in increased levels of plasma triglycerides, total cholesterol, and phospholipids accompanied by a marked reduction in HDL-cholesterol levels compared with non-Tg littermates. Analysis of lipoprotein fractions showed that hyperlipidemia exhibited by Tg rabbits was caused by elevated levels of very-low-density lipoproteins (VLDL) and intermediate-density lipoproteins. Furthermore, postheparin lipoprotein lipase activity significantly decreased in Tg rabbits compared with non-Tg rabbits. Conclusions—These results indicate that apoAII plays an important role in both VLDL and HDL metabolism, possibly through the inhibition of lipoprotein lipase activity. ApoAII Tg rabbits may become a new model for the study of human familial combined hyperlipidemia.

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.