Expression dynamics of Crry at the implantation sites in normal pregnancy and response against miscarriage induction.

In mammals, immune tolerance against fetal tissue has been established for normal pregnancy progression. It is known that Crry regulates complemental activity to prevent injury of the mouse embryo and extra-embryonic tissue. This study aimed to examine the expression appearance and normal localization sites of Crry in the mouse placenta. Also, the emergency responses of Crry were verified at the time of experimental miscarriage induction. Moreover, we investigated an existing similar protein of Crry in animal placentas other than mice. Crry expression level showed a peak at day 8.5 of pregnancy. Trophoblast giant cells and decidual cells indicated a positive reaction to anti-Crry antibody. After treatments of interferon-γ, Crry expression was increased significantly in the survived implantation sites as compared with the controls. However, there was no significant difference in the miscarriage-initiated sites. It disclosed that Crry distributes from the early to middle periods of the placentas and involves complement regulation at the extraembryonic tissue and decidua basalis. Crry also showed an ability to respond to risk against external initiation for urgent miscarriage. Finally, we found anti-mouse Crry antibody-bound proteins in the placenta of many animals. It suggests a potency of Crry to make an environment of immune tolerance in many types of mammal placentas.

Biological potentials for a family of disintegrin and metalloproteinase (ADAMDEC)-1 in mouse normal pregnancy.

Our previous research has indicated local expression of ADAMDEC-1, a family of disintegrin and metalloproteinase, was confirmed in the mouse placentas and enhancement was found in the sites for spontaneous abortion. Present study was aimed to identify biological effects of ADAMDEC-1 in pregnancy process. Syngeneic pairs of C57BL/6J mice and heterogenic mating pairs of CBA/J and DBA/2 mice were used. Pregnant mice were treated with recombinant ADAMDEC-1 protein. Vasculogenesis effects was evaluated using the Matrigel plugs including vascular endothelial growth factor singularity or combination with ADAMDEC-1. ADAMDEC-1 single effects were evaluated by tubal formation and proliferation assays using HuEht-1 endothelial cells. Expression of ADAMDEC-1 was not exactly corresponded with the time periods for miscarriage initiation. ADAMDEC-1 was distributed in normal placentas and fetuses, especially at extraembryonic ectoderm, decidua cells, uterine natural killer (uNK) cells in decidua, trophoblasts in labyrinthine zone, and hematopoietic cells in umbilical blood and fetal liver. ADAMDEC-1 treatment did not affect reproductive performances, while it elevated uNK cell recruitment in placenta and enlarged lumen sizes of the intraplacental vessels. In vitro analysis also indicated ADAMDEC-1 promoting effect on tubal formation and cell length of HuEht-1. qPCR analysis showed that ADAMDEC-1 modified placental gene expression especially for linkage of actin filament rearrangement. Our findings suggested that ADAMDEC-1 is correlated on cell shape, stability, and movement via modification of actin cytoskeleton. ADMADEC-1 suspected to regulate cellular activity of endothelial cells, trophoblasts, and uNK cells and may support normal developing of mouse placentas.

Identification of novel serum markers for the progression of coronary atherosclerosis in WHHLMI rabbits, an animal model of familial hypercholesterolemia.

BACKGROUND AND AIMS: The development of serum markers specific for coronary lesions is important to prevent coronary events. However, analyses of serum markers in humans are affected by environmental factors and non-target diseases. Using an appropriate model animal can reduce these effects. To identify specific markers for coronary atherosclerosis, we comprehensively analyzed the serum of WHHLMI rabbits, which spontaneously develop coronary atherosclerosis. METHODS: Female WHHLMI rabbits were fed standard chow. Serum and plasma were collected under fasting at intervals of 4 months from 4 months old, and a total of 313 lipid molecules, 59 metabolites, lipoprotein lipid levels, and various plasma biochemical parameters were analyzed. The severity of coronary lesions was evaluated with cross-sectional narrowing (CSN) corrected with a frequency of 75%-89% CSN and CSN> 90%. RESULTS: There was a large variation in the severity of coronary lesions in WHHLMI rabbits despite almost no differences in plasma biochemical parameters and aortic lesion area between rabbits with severe and mild coronary lesions. The metabolites and lipid molecules selected as serum markers for coronary atherosclerosis were lysophosphatidylcholine (LPC) 22:4 and diacylglycerol 18:0-18:0 at 4 months old, LPC 20:4 (sn-2), ceramide d18:1-18:2, citric acid plus isocitric acid, and pyroglutamic acid at 8 months old, and phosphatidylethanolamine plasminogen 16:1p-22:2 at 16 months old. CONCLUSIONS: These serum markers were coronary lesion-specific markers independent of cholesterol levels and aortic lesions and may be useful to detect patients who develop cardiovascular disease.

Morphological analyses of the retinal photoreceptor cells in the nocturnally adapted owl monkeys.

Owl monkeys are the only one species possessing the nocturnal lifestyles among the simian monkeys. Their eyes and retinas have been interested associating with the nocturnal adaptation. We examined the cellular specificity and electroretinogram (ERG) reactivity in the retina of the owl monkeys by comparison with the squirrel monkeys, taxonomically close-species and expressing diurnal behavior. Owl monkeys did not have clear structure of the foveal pit by the funduscope, whereas the retinal wholemount specimens indicated a small-condensed spot of the ganglion cells. There were abundant numbers of the rod photoreceptor cells in owl monkeys than those of the squirrel monkeys. However, the owl monkeys' retina did not possess superiority for rod cell-reactivity in the scotopic ERG responses. Scanning electron microscopic observation revealed that the rod cells in owl monkeys' retina had very small-sized inner and outer segments as compared with squirrel monkeys. Owl monkeys showed typical nocturnal traits such as rod-cell dominance. However, the individual photoreceptor cells seemed to be functionally weak for visual capacity, caused from the morphological immaturity at the inner and outer segments.

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.

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.

Nutrient starvation affects expression of LC3 family at the feto-maternal interface during murine placentation.

LC3 - the mammalian homolog of Atg8 - was found as autophagosome membrane binding protein in mammals and widely used as an autophagosomal marker. LC3A, B and C show different expression patterns in each tissue. The aim of this study was to reveal the differences of expression patterns among LC3 families in mouse placenta under normal condition and nutrient starving condition. LC3A and B were highly expressed in decidual cells. LC3A and B were increased in D14 compared with D12 and D16 in mouse placenta, while LC3C was decreased. Starvation induced increase in LC3B expression specifically. Immunohistochemistry showed different expression patterns among LC3A, B and C. LC3A expression in syncytiotrophoblast was vanished by starvation. The results of real time RT-PCR suggested differences between D12 and D16 in autophagic cascade induced by starvation. Taken together, this study suggests that autophagy could play a role in placental invasion system and that nutrient starvation affects LC3B expression.

Dynamics and reproductive effects of complement factors in the spontaneous abortion model of CBA/J×DBA/2 mice.

The complement system is one component of innate immunity that could participate in fetal loss. We have already reported that adipsin, a complement activator in the alternative pathway, is stably expressed in the placenta and that an increase in this expression is related to spontaneous abortion. However, complement inhibitor Crry was concurrently expressed in the placenta, and the role of complement factors during pregnancy was not clear. In the present study, we examined the endogenous regulation of complement factors in placenta and serum by using another model mouse for spontaneous abortion and studied the effect of exogenous complement disruption on pregnancy. Compared to control mice, the CBA/J×DBA/2 model mice had higher expression levels of adipsin in the placenta and serum. Adipsin and complement C3 were localized in the metrial gland and labyrinth regions, and both positive reactive ranges were limited in the maternal blood current in normal implantation sites. These results suggest that extrauterine adipsin hematogenously reaches the placenta, activates complement C3, and promotes destruction of the feto-maternal barrier in aborted implantation sites. Crry was consistently expressed in the placenta and serum and reduced in the resorption sites of CBA/J×DBA/2 mice as compared to normal sites. Injection of recombinant adipsin increased the resorption rate and changed the expression of Th-type cytokines toward a Th1 bias. The present study indicates that adipsin could induce the fetal loss that accompanies the Th1 bias and may be a crucial cause of spontaneous abortion. In addition, the local expression of Crry prevents complement activation in placenta in response to a systemic increase of adipsin.

Influence of atopic dermatitis on reproduction and uterine natural killer cells.

The causal relationship between severe allergic conditions and successful pregnancy remains unclear. We aimed to evaluate reproductive performance in an experimental mouse model of atopic disease (AD), and the appearance of uterine natural killer (uNK) cells that have crucial roles in placental formation was examined. In the NC/Nga pregnant mice with moderate skin allergic lesions and an 8.6-fold elevation of plasma IgE, significant differences were not detected in the reproductive indices of the number of normal fetuses, abortion rate and placental size. There were few uNK cells in the placenta of AD mice, and they showed a significant decrease regarding the immature subtype as compared with controls. These findings revealed that AD disturbs uNK cell differentiation and provides disadvantageous effects on placental formation, although it does not arrest the pregnancy process. It may be possible that specific immunological conditions behind AD operate favorably to recover the reproductive performance.

Characteristic patterns of maternal and fetal arterial construction in the rabbit placenta.

We studied vascular structure of the rabbit placenta, especially on three-dimensional morphological patterns and developmental process. Basic structure of maternal arterial system was re-constructed during day 13-18 of pregnancy, forming main routes for blood supply through the arterial sinuses and radial arteries. Intra-villous spaces were drastically developed showing as branches from the terminal radial arteries. Fetal arterial system was generated accompanied with maternal vascular development, showing characteristic features such as the perforating linear artery, hairpin flexion, and circular anastomoses in the capillaries. From the correlation of maternal and fetal blood currents, gas-exchange style in the rabbit placenta was considered as counter-current and pool mixed patterns. These data demonstrated an original feature for the placental arterial systems in rabbits, which differed from other animals having a property for discoid placenta.

Vasospasm of atherosclerotic coronary arteries precipitates acute ischemic myocardial damage in myocardial infarction-prone strain of the Watanabe heritable hyperlipidemic rabbits.

OBJECTIVE: This study tested the hypothesis that vasospasm can trigger coronary plaque injury and acute ischemic myocardial damage. APPROACH AND RESULTS: Myocardial infarction-prone strain of the Watanabe heritable hyperlipidemic rabbits received an intravenous bolus of ergonovine maleate (0.45 µmol/kg) during intravenous infusion of norepinephrine (12 nmol/kg per minute) to provoke coronary spasm in vivo. After this treatment, coronary angiography demonstrated vasospasm, and the ECG showed ischemic abnormalities (ST depression/elevation and T-wave inversion) in 77% of animals (23/30). These changes normalized after nitroglycerin injection. In rabbits that demonstrated these ECG findings for >20 minutes, echocardiograms showed left ventricular wall motion abnormality. Serum levels of heart-type fatty acid-binding protein, cardiac troponin-I, and myoglobin increased markedly 4 hours after spasm provocation. In coronary lesions of myocardial infarction-prone strain of the Watanabe heritable hyperlipidemic rabbits with provoked coronary spasm, we observed intimal injury in 60.9% in the form of endothelial cell protrusions (39.1%), denudation (30.4%), and macrophage extravasation (56.5%). Plaque disruption with luminal thrombus, however, was only seen in 2 of 23 animals (8.7%), and mural microthrombus was rarely observed (4.3%). CONCLUSIONS: These observations show that provocation of vasospasm in myocardial infarction-prone strain of the Watanabe heritable hyperlipidemic rabbits associates with subsequent ischemic myocardial damage. Although treatment with spasmogens altered aspects of plaque morphology, for example, endothelial protrusion and macrophage emigration, thrombosis was rare in these animals with chronic atherosclerotic disease.

Electrocardiograms corresponding to the development of myocardial infarction in anesthetized WHHLMI rabbits (Oryctolagus cuniculus), an animal model for familial hypercholesterolemia.

The aim of this study was to determine whether features indicative of myocardial ischemia occur in the electrocardiograms (ECG) in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for human familial hypercholesterolemia. ECG were recorded in 110 anesthetized WHHLMI rabbits (age, 10 to 39 mo) by using unipolar and bipolar limb leads with or without chest leads. We noted the following electrocardiographic changes: T wave inversion (37.4%), ST segment depression (31.8%), deep Q wave (16.3%), reduced R wave amplitude (7.3%), ST segment elevation (2.7%), and high T wave (1.8%). These ECG changes resembled those in human patients with coronary heart disease. Histopathologic examination revealed that the left ventricular wall showed acute myocardial lesions, including loss of cross-striations, vacuolar degeneration, coagulation necrosis of cardiac myocytes, and edema between myofibrils, in addition to chronic myocardial lesions such as myocardial fibrosis. The coronary arteries that caused these ECG changes were severely stenosed due to atherosclerotic lesions. Ischemic ECG changes corresponded to the locations of the myocardial lesions. Normal ECG waveforms were similar between WHHLMI rabbits and humans, in contrast to the large differences between rabbits and mice or rats. In conclusion, ischemic ECG changes in WHHLMI rabbits reflect the location of myocardial lesions, making this model useful for studying coronary heart disease.

Myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits with mesenteric fat accumulation are a novel animal model for metabolic syndrome.

OBJECTIVES: To examine whether the myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbit with visceral fat accumulation is a new animal model for human metabolic syndrome, we examined the relationship between mesenteric fat accumulation and insulin resistance, hyperlipidemia and atherosclerosis. METHODS: Glucose tolerance tests were performed using adult (11- to 15-month-old) and middle-aged (17- to 21-month-old) WHHLMI rabbits fed standard chow restrictedly. In addition, lipoprotein lipid levels, serum C-reactive protein (CRP) levels, mesenteric fat weight and physical and physiological parameters were measured. Mesenteric fat was stained immunohistochemically. RESULTS: The mesenteric adipose tissue was positive for monoclonal antibodies against macrophages, C-reactive protein and monocyte chemoattractant protein. In adult rabbits, mesenteric fat correlated to aortic lesion area, insulin resistance, fasting immunoreactive insulin, serum CRP, abdominal circumference and body weight. In middle-aged rabbits, mesenteric fat correlated to lipoprotein lipid levels in addition to the parameters showing a significant correlation in adult rabbits, excluding aortic lesion area. CONCLUSIONS: The WHHLMI rabbit with visceral fat accumulation is a new animal model for metabolic syndrome.