Atherosclerosis In Japanese Quail Research Articles

Antiatherosclerotic Function of Kokuto, Okinawan Noncentrifugal Cane Sugar

In the present study, we investigated the effect of phenolic compounds (PCs) and policosanol of Kokuto, Okinawan noncentrifugal cane sugar, on the development of atherosclerosis. A total of 67 male Japanese quail were divided into eight dietary groups in trial 1. The dietary groups were fed the atherosclerotic diet (AD) containing 5% corn oil, 2% cholesterol, and 30% sucrose or seven different types of Kokuto. Dietary intakes of Kokuto notably prevented the development of atherosclerosis. Furthermore, there was a significant negative correlation between the serum radical scavenging activity and the degree of atherosclerosis in the dietary groups. In trial 2, a total of 63 Japanese quail were fed AD with sucrose, Kokuto, PC extracts from Kokuto, wax extracts from sugar cane, octacosanol, vitamin C, and vitamin E. As a result, the supplementation of the diet with Kokuto and PCs significantly reduced the development of atherosclerosis as compared with the ingestion of AD with sucrose. In conclusion, these findings suggest that, among various components of Kokuto, PCs play a central role for the prevention of experimental atherosclerosis in Japanese quail.

Expression of Cell-Cycle-Regulating Genes in the Development of Atherosclerosis in Japanese Quail (Coturnix japonica)

The levels of mRNA expression in regulatory genes that are involved in the pathological changes of aortic atherosclerotic and fibroblastic intimal thickening was investigated in Japanese quail. The quail were divided into a control diet group and an atherogenic diet group. The quail were euthanized at 2, 4, 8, and 12 wk after consuming either a control diet or an atherogenic diet. Thereafter, both histological and immunohistochemical studies and mRNA expression analysis of the cell-cycle-regulating genes in aortic atherosclerotic lesions were performed on selected ascending aortas and their large branches. In the atherogenic diet group, aortic lipid-containing intimal and atheromatous lesions were seen mainly at 8 and 12 wk, respectively. Semiquantitative reverse-transcription PCR was used to analyze the alterations of mRNA expression on the development of atherosclerotic lesions. Messenger RNA expression of the c-fos and c-src genes showed peak levels at 8 wk in the atherogenic diet group. However, no significant alteration of c-jun mRNA expression was noted during the entire experimental period. According to the progression of aortic atherosclerotic lesions, c-myc mRNA expression in the atherogenic diet group increased chronologically, and the highest level was observed at 12 wk. Alterations in mRNA expression of proliferating cell nuclear antigen and the p27 gene were similar to that of c-myc. The levels of c-myc, proliferating cell nuclear antigen, and p27 mRNA expression was significantly correlated with the degree of aortic atherosclerotic lesion development at 12 wk in our experiment.

Effect of Kokuto, a Non-Centrifugal Cane Sugar, on the Development of Experimental Atherosclerosis in Japanese Quail and Apolipoprotein E Deficient Mice

Kokuto, a traditional cane sugar of Okinawa, has been reported to have antioxidative and lipid-lowering properties. In this experiment, we investigated the effect of three different kinds of Kokuto (KA, KB, and KC) on atherosclerosis in two different animal models : Japanese quail and apolipoprotein E deficient (apoE-/-) mice. Ingestion of Kokuto had no significant effect on the serum and liver lipid levels of Japanese quail. Dietary intake of atherogenic diet (AD) with KA and KB decreased the liver triglyceride level and body weight in apoE-/- mice. Quail fed on AD with KA developed less extent of lipid-containing aortic intimal thickening lesions than those fed on AD with sucrose. Dietary intake of AD with Kokuto or sucrose induced aortic atheromatous lesions in mice, but the extent of atheromatous lesions was roughly comparable between these dietary groups of apoE-/- mice. The present study suggests that Kokuto prevents lipid-containing aortic intimal thickening lesions in Japanese quail.

Temporal and Spatial Localization of Proteoglycan Decorin Transcripts During the Progression of Cholesterol-Induced Atherosclerosis in Japanese Quail

The temporal and spatial distribution of decorin transcripts, endothelial cells, and smooth muscle actin-producing cells were determined during the progression of atherosclerosis in the dorsal aortas of Japanese quail selected for cholesterol-induced atherosclerosis. The quail were placed on either a control diet or a diet containing 0.5% added cholesterol at approximately 16 weeks of age. Dorsal aortas were collected at 2-week intervals for 18 weeks after initiating cholesterol feeding. In situ hybridization for decorin showed the presence of decorin transcripts in the dorsal aorta intima of cholesterol-fed birds beginning at 6 weeks and continuing through the duration of the study. However, in the control-fed birds, decorin transcripts were not found in the intima but were prominent in the adventitia. In cholesterol-fed birds, immunohistochemical localization for endothelial cells showed that intima areas positive for decorin transcripts colocalized with both the endothelial cells and smooth muscle cell layers. In contrast in the control-fed birds, decorin transcripts aligned with the smooth muscle cell layers and not the endothelial cells. These results are suggestive of a potential role for endothelial cells in intimal decorin expression during atherosclerotic plaque formation.

Temporal and Spatial Localization of Proteoglycan Decorin Transcripts During the Progression of Cholesterol-Induced Atherosclerosis in Japanese Quail

The temporal and spatial distribution of decorin transcripts, endothelial cells, and smooth muscle actin-producing cells were determined during the progression of atherosclerosis in the dorsal aortas of Japanese quail selected for cholesterol-induced atherosclerosis. The quail were placed on either a control diet or a diet containing 0.5% added cholesterol at approximately 16 weeks of age. Dorsal aortas were collected at 2-week intervals for 18 weeks after initiating cholesterol feeding. In situ hybridization for decorin showed the presence of decorin transcripts in the dorsal aorta intima of cholesterol-fed birds beginning at 6 weeks and continuing through the duration of the study. However, in the control-fed birds, decorin transcripts were not found in the intima but were prominent in the adventitia. In cholesterol-fed birds, immunohistochemical localization for endothelial cells showed that intima areas positive for decorin transcripts colocalized with both the endothelial cells and smooth muscle cell layers. In contrast in the control-fed birds, decorin transcripts aligned with the smooth muscle cell layers and not the endothelial cells. These results are suggestive of a potential role for endothelial cells in intimal decorin expression during atherosclerotic plaque formation.

Collagen characteristics and organization during the progression of cholesterol-induced atherosclerosis in Japanese quail.

This study reports the concentration of collagen and its hydroxypyridinoline crosslinks, collagen fibril organization in the dorsal aortas, and systolic blood pressure during the progression of atherosclerosis in Japanese quail selected for cholesterol-induced atherosclerosis. The quail were placed on either a control or 0.5% cholesterol-added diet at approximately 16 weeks of age. The concentration of total collagen did not change in the control arteries during the course of the study, whereas at 5 and 10 weeks of cholesterol feeding, collagen levels decreased in the cholesterol-fed birds. Hydroxypyridinoline concentration increased during the duration of the study in the cholesterol-fed birds and by 15 and 20 weeks of cholesterol feeding, levels were significantly increased over those observed in the control arteries. Transmission electron microscopy showed changes in the organization of collagen fibrils. Increased systolic blood pressure was noted beginning at 10 weeks of cholesterol feeding, which is suggestive of other systemic changes induced by hypercholesterolemia. These results demonstrated remodeling of the collagen component of the dorsal aorta extracellular matrix during the progression of atherosclerosis and are suggestive of other systemic cardiovascular system changes.

Expression and localization of the proteoglycan decorin during the progression of cholesterol induced atherosclerosis in Japanese quail: implications for interaction with collagen type I and lipoproteins

The temporal and spatial distribution, and relative levels of the proteoglycan decorin and collagen type I were examined during the progression of atherosclerosis in the dorsal aortas of Japanese quail selected for cholesterol induced atherosclerosis. The quail were placed on either a control or 0.5% added cholesterol diet at approximately 16 weeks of age. Dorsal aortas were collected at 1- or 2-week intervals over a 15-week period after initiating cholesterol feeding. Biochemical analysis for decorin and collagen type I showed that both increased in the cholesterol-fed birds compared to control-fed birds beginning at 9 weeks and continued through the duration of the study. Through immunohistochemical staining for decorin and collagen type I, the spatial localization of decorin and collagen type I in control and less severe plaques in cholesterol-fed birds was most prominent in the arterial adventitia. However, in severe atherosclerotic plaques, decorin was localized in foam cell regions and collagen type I was found surrounding the foam cell regions where decorin accumulated. These results demonstrated a localization of decorin in the core of the atherosclerotic plaque foam cell region with collagen type I being located on the plaque surface.

Epitopes for chicken monoclonal antibodies in spleens of selected Japanese quail lines

A line of Japanese quail selected for high plasma cholesterol is highly susceptible to diet-induced atherosclerosis. Lymphocyte epitopes recognized by mouse anti-chicken monoclonal antibodies (c-mAb), TCR-1, TCR-2, TCR-3, CD-3, CD-4, CD-8, and BU-1a/b were reacted with spleens from quail selected for high (HL) and low (LL) plasma total cholesterol and their nonselected controls (CL). Cross reactivity to c-mAb and effect of line and gender were immunohistochemically evaluated. Chicken spleens were positive controls. Quail were immunologically stimulated with either sheep red blood cells (SRBC) or Brucella abortus 2 weeks before spleens were removed. Quail spleen epitopes of all lines recognized TCR-3 and CD-8 c-mAb, but no other c-mAb. Number of reacting cells and staining intensity to the TCR-3 c-mAb were greater in the HL than in the LL regardless of the stimulating Ag or dose used. For the CD-8 c-mAb, there were no differences among lines in birds receiving SRBC. In B. abortus-immunized birds, sex×line interactions indicated that males of the HL and CL had lower responses than females but LL males were not different than females. TCR-3 and CD8 c-mAb may be useful in studying immunological mechanisms for atherosclerosis in Japanese quail.

Changes in distribution of glycosaminoglycans during the progression of cholesterol induced atherosclerosis in Japanese quail

The temporal and spatial distribution and relative concentration of the proteoglycan glycosaminoglycan component were studied during the progression of atherosclerosis in the systemic arteries of Japanese quail selected for cholesterol induced atherosclerosis (CIA). The CIA quail were placed on either control or 0.5% added cholesterol diets at 3 months of age. The major systemic arteries (dorsal aorta, right and left brachiocephalic) were collected at 1- or 2-week intervals over the 10-week period of cholesterol feeding. In the cholesterol fed quail, alcian blue staining of the dorsal aorta showed elevations of glycosaminoglycans in regions of the artery with atherosclerotic plaque, beginning at the 6-week time point. By biochemical analysis, increases in glycosaminoglycan relative concentration was detected at the 10-week time point. In addition to the change in glycosaminoglycan relative concentration and distribution, the cholesterol fed animals also formed foam cells characteristic of atherosclerotic plaques. Therefore, the conclusion reached was that the CIA line of Japanese quail is a valid animal model for the study of alterations in proteoglycan metabolism in atherosclerotic plaques induced by hypercholesterolemia.

Dietary Effects of Corn Oil, Oleic Acid, Perilla Oil, and Primrose Oil on Plasma and Hepatic Lipid Level and Atherosclerosis in Japanese Quail.

To compare the atherogenecity of different fats and oils, a total of forty, 40-day-old male Japanese quails were fed one of the following diets for three months: basal diet (control), a diet-containing 15% corn oil (CO) and 2% cholesterol (CH), a diet-containing 15% oleic acid (OL) and 2% CH, a diet-containing 15% perilla oil (PE) and 2% CH, a diet-containing 15% evening [corrected] primrose oil (PR) and 2% CH. A higher plasma cholesterol concentration was found in the birds in the CO and OL groups, whereas the PE and PR groups showed a much lower level of plasma cholesterol than the CO and OL groups. In proportion to the increased plasma cholesterol, both CO and OL groups showed narrowing of the lumen of the ascending aorta and its large branches due to marked lipid-rich intimal thickening. Ultrastructural changes in the ascending aorta and its large branches were correlated with the degree of intimal thickening. The major foam cell types were macrophages and fibroblastic cells. The PE and PR groups showed the fewest lipid-rich intimal thickening lesions in their ascending aorta and its large branches. These findings suggest that the alpha-linolenic acid contained in perilla oil is less atherogenic than oleic and linoleic acid, and gamma-linolenic acid contained in evening [corrected] primrose oil has a tendency to decrease the plasma lipid level.

Immunocytochemical and Scanning Electron Microscopic Studies of Atherosclerosis in Japanese Quail

The major cellular components of atherosclerotic lesions in several species have been shown to be smooth muscle cells (SMC) and macrophages. Many studies suggest the composition of a lesion varies depending on the stage of lesion development. For example, macrophages are believed to be involved in the initial events of fatty streak formation in many animals. This communication presents the first cellular study of quail atherosclerosis and demonstrates the alteration of cellular structure during the process of the disease in quail fed a cholesterol diet. Monoclonal antibodies to alpha-actin and chicken macrophages effectively identified the presence of SMC and macrophages, respectively, as constituents of the atherosclerotic lesions. Macrophage presence, as well as SMC proliferation, was observed in early lesions. Although the first cell type to be involved in the initial stages of atherogenesis cannot be defined, the results suggest early intervention of macrophages and SMC. Scanning electron microscopic examination of the aortic arch demonstrates the obvious differences in appearance of the endothelial surface of normal and diseased quail. The accumulation of subendothelial foam cells causes the lumen surface to bulge irregularly into the lumen. The results of the present study are important to the evaluation of the key cellular events of atherogenesis.

実験的ウズラ動脈硬化症における電顕および組織化学的観察

A marked plaque was produced at the tunica intima of the ascending aorta in all of the Japanese quails of 9 weeks old which fed on atherogenic diet containing 2% cholesterol for 8 weeks, while no structural changes of aortic wall were observed in Japanese quails which fed on normal basic food for the same period. The media of aorta in normal quails consist of smooth muscle cells, myofibroblast-like cells (MF) cells), and many successive elastic membranes. At the atherosclerotic lesion, many MF cells migrated from media into intima, and a part of smooth muscle cells were also differentiated to MF cells. Moreover, the most migrating MF cells differentiated to foam cells at the intimal thickness regions, and a few other MF cells also differentiated into endothelial cells of newly forming capillaries. By the immunohistochemical stainings, medial smooth muscle cells were negatively stained with anti-vimentin antibody, and the majority of cells in the intima (MF cells, foam cells, and endothelial cells) contained vimentin filaments. These results indicate that MF cells play a very important role in the development of atherosclerosis in Japanese quail. The morphologicals study offers some new insights into the evaluation of Japanese quails as an animal model of atherosclerosis.

Effect of snake venom of Agkistrodon halys on atherosclerosis and blood characteristics in Japanese quail

Extracts of snake venom have been widely used for the treatment of vascular thrombotic diseases, yet the therapeutic mechanism is not clear. The effect of snake venom fractions on atherosclerosis in Japanese quail was studied. The venom of Agkistrodon halys was fractionated by DEAE-cellulose chromatography and the pooled protein fractions that resulted were injected intravenously into the quail with aortic atherosclerosis induced by dietary cholesterol. After 7 weeks of injections on every other day, the quail were killed, blood clotting times and serum cholesterol levels were determined, and aortic atherosclerosis and fatty liver were scored. The results showed that while no regression of atherosclerosis was observed, the lowering of serum cholesterol, prolonged blood clotting time and reduced fatty liver were significantly affected by the injection of one of the pooled protein fractions. This venom fraction contained two major protein components, one of which had arginine esterase activity. From this study we conclude that snake venom has little effect on the regression of atherosclerosis, but it prolongs blood clotting and lowers serum cholesterol.

Prevention of hypercholesterolemia and atherosclerosis in Japanese quail by high intake of soy protein

This study was undertaken to determine the influence of levels of soy protein on cholesterol metabolism and the development of atherosclerosis in Japanese quail ( Coturnix coturnix japonica). Quail were fed purified diets containing one of four levels (10, 20, 40 or 60%) of soy protein either with (atherogenic) or without (control) 0.5% cholesterol. Results showed that higher proportions of protein (40 and 60%) in atherogenic diets had a preventive effect on the development of atherosclerosis in adult male quail. For the atherogenic diets the higher protein levels resulted in significantly lower serum and aorta cholesterol concentrations than observed with the 10 or 20% protein levels and free of aortic atherosclerosis. A dual isotope technique was used to measure the absorption rates for cholesterol in quail fed with 20 and 40% soy protein. Absorption rates were not affected by the level of dietary protein but were influenced by the presence of dietary cholesterol. Relative rates were 40% in quail fed control diets and 30% in quail fed atherogenic diets. The excretion of neutral sterols, largely cholesterol, and bile acids increased with the high intake of soy protein. These results demonstrate that in quail the presence of higher dietary levels of soy protein has both a hypocholesterolemic action and a preventive effect on cholesterol-induced atherosclerosis and one of the possible mechanisms is through increased excretion of cholesterol.

The effect of dietary cholesterol level on lipid status and initiation of atherosclerosis in Japanese quail

The effect of variations in dietary cholesterol level on the development of hyperlipidemia, tissue cholesterol status and incidence of atherosclerosis was investigated in male Japanese quail of strain SEA (Susceptible to Experimental Atherosclerosis). Groups of 12, five-week-old, birds were fed one of four purified diets. The basal diet contained (weight %): soyprotein, 35; beef tallow, 10; glucose, 46; three atherogenic diets were prepared by replacing glucose with 0.25, 0.5 and 1.0% cholesterol in the basal diet formula. After 4 weeks, birds were killed and serum, thoracic aortae, brachiocephalic arteries, and liver were taken for analysis. Serum cholesterol was increased (relative to basal values) by 15, 127 and 229% for birds fed the 0.25, 0.5 and 1.0% cholesterol diets respectively; increases were due to elevations of cholesterol in the very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions. Hepatic and arterial cholesterol levels increased with increasing dietary cholesterol levels, but there were significant, between-group differences in the case of liver cholesterol only. Lesion incidence was 0, 8, 43 and 50% for birds fed the basal, 0.25, 0.50 and 1.0% cholesterol diets respectively. The small dot-like, yellow lesions were found predominantly in the right brachiocephalic artery and to a lesser extent in the thoracic aorta; no lesions were found in the left brachiocephalic artery. The extreme rapidity with which Japanese quail of strain SEA develop early atherosclerotic lesions makes them an attractive animal model for studying the early development of atherosclerosis in man.

Dietary Induction of Hypercholesterolemia and Atherosclerosis in Japanese Quail of Strain SEA

Groups of 12 male Japanese quail (Coturnix coturnix japonica) of a strain susceptible to experimental atherosclerosis (SEA) were fed one of three purified diets (one basal and two atherogenic) for 8 wk. The basal diet contained: glucose (46%), soy protein (35%) and beef tallow (10%). Atherogenic diets were prepared by replacing glucose with either 1% cholesterol or 1% cholesterol plus 0.5% cholic acid. Both atherogenic diets induced hypercholesterolemia, with serum cholesterol increased 202 and 336% above basal values for birds fed the cholesterol and cholesterol/cholic acid diets, respectively. The hypercholesterolemia was characterized by an increase in serum levels of very low density and low density lipoprotein cholesterol. No atherosclerotic lesions were found in birds fed the basal diet, but lesion incidence in the dorsal aorta and brachiocephalic arteries was 75 and 100% for birds fed the cholesterol and cholesterol/cholic acid diets, respectively. Arterial scores (percentage of arterial surface area covered by lesions), arterial cholesterol concentration and total liver cholesterol were significantly higher (P < 0.05) for birds fed the cholic acid containing diet than for those fed the diet having cholesterol only. Severity of atherosclerotic score and arterial cholesterol concentration were positively correlated (P < 0.01 in both cases) with serum cholesterol concentration. The two indices of the severity of atherosclerosis (arterial score and arterial cholesterol concentration) were highly and linearly correlated (r = 0.88, P < 0.01). The SEA strain of quail appears to be a promising model for the study of atherosclerosis. However, more research on the nature of the atherosclerotic lesions, mechanisms of lesion development and response to nutritional manipulations is needed before results can be extrapolated to humans.

Temporal association between arterial cholesterol deposition, thymidine incorporation into DNA, and atherosclerosis in Japanese quail fed an atherogenic diet

Male Japanese quail (strain SEA) rapidly develop atherosclerotic lesions in the aorta and brachiocephalic arteries when fed an atherogenic diet containing 1.0% cholesterol and 0.5% cholic acid. The present study was conducted to determine time parameters of the atherosclerotic response. Groups of 20 quail fed the atherogenic diet were killed at 0 days, 1 day, 3 days, or weekly from 1 to 12 weeks. Quail fed the atherogenic diet for 1 day showed a significant increase in serum cholesterol; a plateau was reached by 2 weeks. A significant increase in arterial cholesterol was seen after 2 weeks on the atherogenic diet, and arterial cholesterol showed a linear increase with time from 2 to 12 weeks. Increased incorporation of tritiated thymidine into the DNA of arterial cells was first seen at 2 weeks; thymidine incorporation increased to a maximum value at 9 weeks, then declined to 50–60% of the 9-week value at weeks 11 and 12. Grossly visible atherosclerotic lesions were first seen at 3 weeks, and 90% of birds showed gross atherosclerotic lesions by 8 weeks. Atherosclerosis induced in Japanese quail by feeding cholesterol and cholic acid is characterized initially by lipid deposition in the arterial wall, followed by increased incorporation of tritiated thymidine and the appearance of gross lesions.

Experimental dietary production of aortic atherosclerosis in Japanese quail

Japanese quail ( Coturnix coturnix japonica) were evaluated as laboratory animals for use in atherosclerosis research by measuring plasma cholesterol along with macroscopic examination of the large arteries for atheromatous lesions. Eighteen-day old quail chicks were placed on a series of 11 varied diets to elicit response to sugar, fats and cholesterol. Birds were sacrificed at the end of 3, 6 and 9 months. Atheromas were found in birds given diets with high saturated fat and added cholesterol. Well defined indications of early stage arterial damage was observed in quail on several of the diets containing cholesterol, high sucrose and high saturated fatty acids (SAFA).