Kazutoshi Nishijima

Human C-Reactive Protein Does Not Promote Atherosclerosis in Transgenic Rabbits

Background— Although there is a statistically significant association between modestly raised baseline plasma C-reactive protein (CRP) values and future cardiovascular events, the debate is still unsettled in regard to whether CRP plays a causal role in the pathogenesis of atherosclerosis. Methods and Results— We generated 2 lines of transgenic (Tg) rabbits expressing human CRP (hCRP). The plasma levels of hCRP in hCRP-Tg-1 and hCRP-Tg-2 rabbits were 0.4±0.13 (n=14) and 57.8±20.6 mg/L (n=12), respectively. In addition, hCRP isolated from Tg rabbit plasma exhibited the ability to activate the rabbit complement. To define the role of hCRP in atherosclerosis, we compared the susceptibility of hCRP-Tg rabbits to cholesterol-rich diet-induced aortic and coronary atherosclerosis with that of non-Tg rabbits. After being fed with a cholesterol-rich diet for 16 weeks, Tg and non-Tg rabbits developed similar hypercholesterolemia and lesion sizes in both aortic and coronary arteries. Immunohistochemical staining and Western blotting revealed that hCRP was indeed present in the lesions but did not affect macrophage accumulation and smooth muscle cell proliferation of the lesions. Conclusions— Neither high nor low plasma concentrations of hCRP affected aortic or coronary atherosclerosis lesion formation in hCRP-Tg rabbits.

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 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.

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit.

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN2) and then preserved in the LN2. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN2. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.

Occlusal tooth wear in male F344/N rats with aging

With the aim of clarifying the aging properties of an animal model, the progress of occlusal tooth wear (OTW) of molars in male F344/N rats was monitored. Dried maxilla and mandible specimens from 61 male F344/N rats, aged 7 to >30 months, were used. The levels of OTW of all molars were monitored with aging. The cuspis dentis of molar teeth were worn out by 7 months (M) of age, and the occlusal surface became flat. As for each molar tooth (M(1-3), numbered in accordance with its position), OTW of M(1) was more severe in the lower than in the upper jaw, whereas M(3) was more severe in the upper than the lower jaw. OTW of M(2) in both the upper and the lower jaws progressed rapidly after 27M. OTW in male F344/N rats progressed faster than in females. However, when compensated for life span, both genders had similar profiles in OTW progress with aging. This study suggested that male rats were more convenient than females as a model for gerodontological research because of the earlier course of OTW progress.

Aging change of mandibular condyle in female F344/N rat.

To ascertain whether laboratory rats represent an adequate animal model for aging oral cavity research, this study focused on the morphology of the mandibular condyle. Aging changes of cartilaginous conformation and shape of the mandibular condyles were analyzed in female F344/N rats. In the condylar cartilage, articular, proliferative cell and hypertrophic cell layers were observed in 1-month-old (M) rat, whereas flattened cell layer was notable at 2M. A mature cell layer was observed in the condylar cartilage of rats at 7.7 M and over. Deranged cartilaginous layers and thinning articular layer were observed in 30.9 M rat. The sagittal length of the condyle decreased, whereas the frontal one increased with aging and/or age. There were three phases in the transition patterns of the size of the condyle, which seemed to correspond to the respective growing, aging and senescence phases in the rat. The results suggested that degenerative change of condylar cartilage in rat was similar to that in human, whereas change of the shape of the condyle was different between rat and human, caused by a different pattern of mastication.

Effects of Cholesterol-Loaded Cyclodextrins on the Rate and the Quality of Motility in Frozen and Thawed Rabbit Sperm

The motility of sperm after freezing and thawing is critical for effective cryopreservation. It is known that supplementation with cholesterol-loaded cyclodextrin (CLC) improves cryosurvival of sperm in various animals. To clarify the effects of supplementation with CLC on rabbit sperm motility after freezing and thawing, rabbit sperm motility was analyzed using a computer-assisted sperm analysis system. Sperm motility with CLC supplementation was 29.4 ± 9.6% (mean ± SD), which was significantly higher than that of controls (20.8 ± 7.1%, P<0.05). The curvilinear velocity of sperm with CLC exceeded that of controls, whereas the values for linearity and wobble were significantly lower in sperm with CLC compared with controls. After artificial insemination, 44.3% of recovered ova were fertilized in the CLC-supplemented group, which was higher than the percentage in the control group (36.4%). The results indicate that supplementation with CLC improves the rate and quality of motility in rabbit sperm after freezing and thawing, and would be advantageous for successful cryopreservation.

Bone degeneration and its recovery in SMP30/GNL-knockout mice

Senescence marker protein-30 (SMP30) decreases androgen-independently with aging and is a lactone-hydrolyzing enzyme gluconolactonase (GNL) that is involved in vitamin C biosynthesis. In the present study, bone properties of SMP30/GNL knockout (KO) mice with deficiency in vitamin C synthesis were investigated to reveal the effects of SMP30/GNL and exogenous vitamin C supplementation on bone formation. Mineral content (BMC) and mineral density (BMD) of the mandible and femur of SMP30/GNL KO and wild-type mice at 2 and 3 months of age with or without vitamin C supplementation were measured by dual-energy X-ray absorptiometry. Body and bone weight of both age groups decreased and became significantly lower than those of wild-type mice. The bones of SMP30/GNL KO mice were rough and porous, with BMC and BMD significantly below wild-type. Oral supplementation with vitamin C eliminated differences in body weight, bone weight, BMC, and BMD between SMP30/GNL KO and wild-type mice at each age. These results indicate that bone degeneration in SMP30/GNL KO mice was caused by lack of vitamin C, and that this mouse strain is an appropriate model for bone metabolism in humans, which have no ability to synthesize vitamin C.

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.

Motility and fertility of rabbit sperm cryopreserved using soybean lecithin as an alternative to egg yolk

This study was conducted to investigate whether soy lecithin can be used as an alternative cryoprotectant to establish a procedure that does not require the use of egg yolk to cryopreserve rabbit strains. Semen from Japanese White rabbits was frozen with HEPES extender containing 20% egg yolk (EYH), 0.5% (Lec-0.5), 1.5% (Lec-1.5), 2.5% (Lec-2.5), or 3.5% (Lec-3.5; wt/vol) lecithin (type IV-S, ≥30%), and the motility of thawed sperm was analyzed. The sperm motility in the Lec-1.5 group was significantly higher than that in the Lec-2.5 and 3.5 groups and equivalent to the EYH group. From 17 rounds of artificial insemination with frozen-thawed sperm in the EYH and Lec-1.5 groups, 12 rabbits in both groups were pregnant (70.6%) and delivered offspring. The litter size was 3.3 in the EYH group and 5.1 in the Lec-1.5 group. These results indicate that soy lecithin can be used as a substitute for egg yolk as a cryoprotectant on the basis of motility and fertility of the frozen-thawed rabbit sperm and that 1.5% lecithin (type IV-S, ≥30%) in the semen extender was the optimum concentration for rabbit sperm cryopreservation.