Salvia miltiorrhiza inhibits intimal hyperplasia and monocyte chemotactic protein-1 expression after balloon injury in cholesterol-fed rabbits

Antioxidants that prevent low density lipoproteins (LDL) from oxidation may inhibit atherosclerosis and post-angioplasty restenosis. Salvia miltiorrhiza (SM) has been shown to inhibit LDL oxidation and reduce atherosclerosis in cholesterol-fed rabbits. The effects of SM on neointimal hyperplasia and monocyte chemotactic protein-1 (MCP-1) expression after balloon injury were studied. Male New Zealand white rabbits were fed a 2% cholesterol diet together with daily SM (4.8 gm/kg body wt.) treatment (SM; n=10) or without SM as a control (C; n=9) for 6 weeks. Probucol-treated (0.6 gm/kg body wt.) rabbits (P; n=9) were used as a positive control group. A balloon injury of the abdominal aorta was performed at the end of the third week. Aortas were harvested at the end of 6 weeks. The plasma cholesterol levels were lowered in SM group. The neointimal hyperplasia in abdominal aortas was significantly inhibited in SM group [neointima/media area ratio: 0.63+/-0.05 (SM) versus 0.78+/-0.05 (C); P < 0.05] and in P group [0.45+/-0.02 (P) versus 0.78+/-0.05 (C); P < 0.05] when compared with C group. SM treatment significantly reduced MCP-1 mRNA and protein expression in balloon-injured abdominal aorta. These inhibitory effects on intimal response after balloon injury might be attributed to antioxidant capacity and cholesterol lowering effect of SM. SM treatment may offer some protection against post-angioplasty restenosis.     

99mTechnetium-labeled low density lipoprotein: receptor recognition and intracellular sequestration of radiolabel

99MTechnetium-labeled low density lipoprotein (99MTc-labeled LDL) was developed to detect atherosclerosis by external imaging with the gamma scintillation camera (Lees, et al. J. Nucl. Med. 1985. 26: 1056-1062; Lees, et al. Arteriosclerosis. 1988. 8: 461-470). The present study examined high affinity LDL receptor recognition and intracellular sequestration of 99MTc-labeled LDL by fibroblasts. There were no significant differences between 99MTc-labeled LDL and 125I-labeled LDL in binding parameters or percent inhibition of accumulation, which indicated that 99MTc labeling did not alter receptor recognition of LDL. At 4 degrees C the Kd (+SE) for 99MTc-labeled LDL and 125I-labeled LDL, respectively, was 1.52 +/- 0.24 and 1.45 +/- 0.14 micrograms/ml; Bmax (+/- SE) was 5.45 +/- 0.48 and 4.89 +/- 0.25 ng/well, respectively. Binding was saturated at about 2 micrograms/ml. The complete linearity of 99MTc-labeled LDL accumulation from 0-6 h and the positive slope from 6-24 h indicated that radiolabel that entered cells as 99MTc-labeled LDL was sequestered; pulse-chase experiments, which measured residual cell-associated radioactivity out to 24 h, also showed that radiolabel was trapped. Because radiolabel sequestration was essentially complete, and because 99MTc-labeled LDL was recognized by the LDL receptor equally as well as 125I-labeled LDL, it should be useful not only for imaging atherosclerosis, but also for quantitatively determining sites of utilization and degradation of LDL.     

17beta-Estradiol but not the phytoestrogen naringenin attenuates aortic cholesterol accumulation in WHHL rabbits

The effects of 17beta-estradiol (17beta-E(2)) or the phytoestrogen naringenin on spontaneous atherosclerosis were studied in 36 ovariectomized homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits receiving a semisynthetic control diet; this diet added 0.0040% 17beta-E(2;) or 0.20% naringenin, for 16 weeks. The uterine weight was increased (P < 0.001) and the concentration of estrogen receptor alpha was decreased (P < 0.001) in the 17beta-E(2) group compared with the controls. Total plasma cholesterol and triglycerides were not different from those in the controls. In lipoproteins, HDL cholesterol was increased (P < 0.01), and LDL triglyceride and IDL triglyceride were lowered (P < 0.05). The oxidation (as concentration of malondialdehyde) was increased in LDL (P < 0.05) but not in plasma. The cholesterol accumulation was decreased (P < 0.05) in the ascending aorta and in the total aorta but the ratio of intima to media and area of intima in ascending, thoracic, and abdominal aorta were not significantly different. In the naringenin group the only differences, compared with the control group, were increased HDL cholesterol (P < 0.001) and decreased activity of glutathione reductase (P < 0.05).In conclusion, 17beta-E(2), but not naringenin, attenuated aortic cholesterol accumulation independently of plasma and LDL cholesterol. Further, these results support previously suggested pro-oxidant ability of 17beta-E(2) toward LDL and a possible connection between the pro-oxidant nature of 17beta-E(2) and its antiatherogenic effect.     

Turnover of 125I-VLDL and 131I-LDL apolipoprotein B in rabbits fed diets containing casein or soy protein

Rabbits fed low-fat, cholesterol-free, semi-purified diets containing casein developed a marked hypercholesterolemia compared to rabbits fed a similar diet containing soy protein (plasma cholesterol 281 +/- 31 vs. 86 +/- 9 mg/dl; P less than 0.05). Turnover studies (three per dietary group) were carried out in which homologous 125I-labeled VLDL and 131I-labeled LDL were injected simultaneously into casein- (n = 8) or soy protein- (n = 9) fed rabbits. ApoB-specific activities were determined in VLDL, IDL and LDL isolated from the pooled plasma of two or three rabbits per dietary group. The production rate of VLDL apoB (1.20 +/- 0.3 vs. 1.09 +/- 0.1 mg/h per kg) was similar for the two dietary groups. The fractional catabolic rate of VLDL apoB was lower for the casein group (0.15 +/- 0.03 vs. 0.23 +/- 0.01.h-1; 0.05 less than P less than 0.10). Although the pool size of VLDL apoB was higher in the casein group (8 +/- 2 vs. 5 +/- 0.3 mg/kg), this value did not reach statistical significance. For LDL apoB, the increased pool size in casein-fed rabbits (30 +/- 5 vs. 5 +/- 1 mg/kg; P less than 0.01) was associated with a decreased fractional catabolic rate (0.03 +/- 0.005 vs. 0.08 +/- 0.008.h-1; P less than 0.01) and a 2-fold increase in the production rate of LDL apoB (1 +/- 0.3 vs. 0.4 +/- 0.06 mg/kg per h; 0.05 less than P less than 0.10) compared to rabbits fed soy protein. Analysis of precursor-product relationships between the various lipoprotein fractions showed that casein-fed rabbits synthesized a higher proportion of LDL apoB (95% +/- 2 vs. 67% +/- 2; P less than 0.001) independent of VLDL catabolism. These results support the concept that the hypercholesterolemia in casein-fed rabbits is associated with impaired LDL removal consistent with a down-regulation of LDL receptors. These changes do not occur when the casein is replaced by soy protein.     

Increased low density lipoprotein degradation in aorta of irradiated mice is inhibited by preenrichment of low density lipoprotein with alpha-tocopherol

We previously reported that upper thoracic exposure to ionizing radiation (IR) accelerates fatty streak formation in C57BL/6 mice and that such effects are inhibited by overexpression of the antioxidant enzyme CuZn-superoxide dismutase (SOD). Notably, IR-accelerated lesion formation is strictly dependent on a high fat diet (i.e., atherogenic lipoproteins) but does not involve alterations in circulating lipid or lipoprotein levels. We thus proposed that IR promotes changes in the artery wall that enhance the deposition of lipoprotein lipids. To address this hypothesis, we examined the effects of IR on aortic accumulation and degradation of low density lipoproteins (LDL). Ten-week-old C57BL/6 mice were exposed to a single (8-Gy) dose of (60)Co radiation to the upper thoracic area or were sham irradiated (controls) and were then placed on the high fat diet. Five days postexposure, the mice received either (125)I-labeled LDL ((125)I-LDL) (which was used to measure intact LDL) or (125)I-labeled tyramine cellobiose ((125)I-TC)-LDL (which was used to measure both intact and cell-degraded LDL) via tail vein injection. On the basis of trichloroacetic acid (TCA)-precipitable counts in retroorbital blood samples, > or =95% of donor LDL was cleared within 24 h and there were no differences in time-averaged plasma concentrations of the two forms of LDL among irradiated and control mice. Aortic values increased markedly within the first hour and thereafter exhibited a slow increase up to 24 h. There were no differences between irradiated and control mice at 1 h, when values primarily reflected LDL entry, but a divergence was observed thereafter. At 24 h, (125)I-TC-associated counts were 1.8-fold higher in irradiated mice (P = 0.10). In contrast, (125)I-LDL-associated counts were 30% lower in irradiated mice (P< 0.05), suggesting that most of the retained (125)I-TC was associated with LDL degradation products. Consistent with the proposed involvement of oxidative or redox-regulated events, IR-induced LDL degradation was lower in SOD-transgenic than wild-type mice (P<0.05). The importance of LDL oxidation was suggested by observations that IR-induced LDL degradation was significantly reduced by preenriching LDL with alpha-tocopherol. On the basis of these results, we propose that IR elicits SOD-inhibitable changes in the artery wall that enhance LDL oxidation and degradation leading to the deposition of LDL-borne lipids. These studies provide additional support for the role of oxidation in lipoprotein lipid deposition and atherogenesis and suggest that IR promotes an arterial environment that stimulates this process in vivo.     

糖尿病和非糖尿病动脉粥样硬化兔模型的建立

目的建立兔动脉粥样硬化和糖尿病动脉粥样硬化模型并比较其动脉粥样硬化病变的特点。方法四氧嘧啶静脉推注诱发糖尿病后,行腹主动脉球囊损伤术拉伤内皮并饲高脂饲料建立糖尿病动脉粥样硬化兔模型,非糖尿病动脉粥样硬化兔模型静脉推注生理盐水,余处理相同。喂养10周做腹主动脉造影和腹主动脉内超声后处死,取腹主动脉横切片做HE染色和免疫组化,比较两组兔主动脉内膜/中膜比值及巨噬细胞、平滑肌细胞含量,以评价动脉粥样硬化病变的程度和性质。结果所有兔胸主动脉粥样硬化病变明显轻于腹主动脉;糖尿病动脉粥样硬化兔腹主动脉壁特别是近血管腔处巨噬细胞浸润明显多于动脉粥样硬化兔,而平滑肌细胞含量显著减少。结论糖尿病动脉粥样硬化兔的腹主动脉粥样硬化病变内有更加活跃的炎症细胞浸润,提示病变性质更加不稳定。     

Paraoxonase activity is reduced by a pro-atherosclerotic diet in rabbits

Serum paraoxonase (PON1) is believed to protect against the development of atherosclerosis because of its ability to retard the oxidation of low-density lipoprotein (LDL) by hydrolysing LDL-associated phospholipid and cholesteryl-ester hydroperoxides. We have examined the relationship between PON1 and atherosclerosis development in transgenic rabbits overexpressing human apolipoprotein (apo) A-I and nontransgenic littermates fed a pro-atherogenic diet. PON1 activity was higher in transgenic (4006.1 +/- 716.7 nmol/min/ml) compared to control (3078.5 +/- 623.3 nmol/min/ml) rabbits (P < 0.01) while high-density lipoprotein (HDL) cholesterol was 1.84 +/- 0.54 mmol/L in transgenic rabbits and 0.57 +/- 0.21 mmol/L in control rabbits (P = 0.0001). After feeding rabbits a high-cholesterol diet for 14 weeks HDL-cholesterol fell by 70% in both transgenic and control rabbits (P < 0.001 compared to week 0) PON1 activity fell by 50% in both groups of rabbits (P < 0. 01 compared to week 0). The amount of thoracic aortic surface area covered by lesions was 29 +/- 16% in the control group and 26 +/- 15% in the transgenic group (P = NS). A pro-atherosclerotic diet reduces PON1 which may exaggerate the effects of the diet on the development of atherosclerosis.     

Regional distribution of glutathione-related antioxidant defences in the normal rabbit aorta

In 6 normal rabbits, the aortic arch, the descending thoracic and the abdominal aorta were tested for non proteic thiol compounds, selenium-dependent and selenium-independent glutatione peroxidase, glutatione reductase, glutatione transferase and thiobarbituric acid reactive substances. The aortic arch showed the greatest content of non proteic thiol compounds and thiobarbituric acid reactive substances, associated to the highest activities of glutathione-related enzymes. However, not significant differences were detectable between aortic arch and descending thoracic aorta, except for the glutathione transferase activity (0.395 +/- 0.031 vs 0.330 +/- 0.053 U/mg protein, p less than 0.05). Furthermore, both aortic arch and descending thoracic aorta showed significantly higher values of non proteic thiol compounds (46.05 +/- 10.15% and 33 +/- 13.5%, p less than 0.05), selenium-dependent glutathione peroxidase activity (70.35 +/- 26% and 54.3 +/- 9.5%, p less than 0.05), glutathione reductase activity (25.4 +/- 7% and 18.4 +/- 4.5%, p less than 0.05) and thiobarbituric acid reactive substances (65.8 +/- 18% and 47.2 +/- 17%, p less than 0.05) with respect to the abdominal aorta. The selenium-independent glutathione peroxidase activity was not detectable. In conclusion, a biochemical gradient in glutathione-related antioxidant defences and thiobarbituric acid reactive substances proceeding from the proximal to the distal segments seems to exist in the normal rabbit aorta. These results could contribute to explain the non homogeneous distribution of experimental atherosclerosis in the rabbit aorta.     

The ligand-binding function of hepatic lipase modulates the development of atherosclerosis in transgenic mice

To investigate the separate contributions of the lipolytic versus ligand-binding function of hepatic lipase (HL) to plasma lipoprotein metabolism and atherosclerosis, we compared mice expressing catalytically active wild-type HL (HL-WT) and inactive HL (HL-S145G) with no endogenous expression of mouse apoE or HL (E-KO x HL-KO, where KO is knockout). HL-WT and HL-S145G reduced plasma cholesterol (by 40 and 57%, respectively), non-high density lipoprotein cholesterol (by 48 and 61%, respectively), and apoB (by 36 and 44%, respectively) (p < 0.01), but only HL-WT decreased high density lipoprotein cholesterol (by 67%) and apoA-I (by 54%). Compared with E-KO x HL-KO mice, both active and inactive HL lowered the pro-atherogenic lipoproteins by enhancing the catabolism of autologous (125)I-apoB very low density/intermediate density lipoprotein (VLDL/IDL) (fractional catabolic rates of 2.87 +/- 0.04/day for E-KO x HL-KO, 3.77 +/- 0.03/day for E-KO x HL-WT, and 3.63 +/- 0.09/day for E-KO x HL-S145G mice) and (125)I-apoB-48 low density lipoprotein (LDL) (fractional catabolic rates of 5.67 +/- 0.34/day for E-KO x HL-KO, 18.88 +/- 1.72/day for E-KO x HL-WT, and 9.01 +/- 0.14/day for E-KO x HL-S145G mice). In contrast, the catabolism of apoE-free, (131)I-apoB-100 LDL was not increased by either HL-WT or HL-S145G. Infusion of the receptor-associated protein (RAP), which blocks LDL receptor-related protein function, decreased plasma clearance and hepatic uptake of (131)I-apoB-48 LDL induced by HL-S145G. Despite their similar effects on lowering pro-atherogenic apoB-containing lipoproteins, HL-WT enhanced atherosclerosis by up to 50%, whereas HL-S145G markedly reduced aortic atherosclerosis by up to 96% (p < 0.02) in both male and female E-KO x HL-KO mice. These data identify a major receptor pathway (LDL receptor-related protein) by which the ligand-binding function of HL alters remnant lipoprotein uptake in vivo and delineate the separate contributions of the lipolytic versus ligand-binding function of HL to plasma lipoprotein size and metabolism, identifying an anti-atherogenic role of the ligand-binding function of HL in vivo.     

Impact of Age-Dependent Adventitia Inflammation on Structural Alteration of Abdominal Aorta in Hyperlipidemic Mice

Background

The adventitia is suggested to contribute to vascular remodeling; however, the site-selective inflammatory responses in association with the development of atherosclerosis remain to be elucidated.

Methods and Results

Wild-type or apolipoprotein E knockout male C57BL/6J background mice were fed standard chow for 16, 32, and 52 weeks, and the morphology of the aortic arch, descending aorta, and abdominal aorta was compared. Atheromatous plaque formation progressed with age, particularly in the aortic arch and abdominal aorta but not in the descending aorta. In addition, we found that the numbers of macrophages, T-lymphocytes, and microvessels, assessed by anti-F4/80, CD3, and CD31 antibodies, were higher in the adventitia of the abdominal aorta at 52 weeks. These numbers were positively correlated with plaque formation, but negatively correlated with elastin content, resulting in the enlargement of the total vessel area. In aortic tissues, interleukin-6 levels increased in the atheromatous plaque with age, whereas the level of regulated on activation, normal T cell expressed and secreted (RANTES) increased with age, and compared with other sites, it was particularly distributed in inflammatory cells in the adventitia of the abdominal aorta.

Conclusion

This study suggests that adventitial inflammation contributes to the age-dependent structural alterations, and that the activation/inactivation of cytokines/chemokines is involved in the process.     

A novel mechanism by which probucol lowers low density lipoprotein levels demonstrated in the LDL receptor-deficient rabbit

Treatment of low density lipoprotein (LDL) receptor-deficient rabbits (WHHL rabbits) with probucol (1% w/w in a chow diet) lowered their LDL-cholesterol levels by 36%, consonant with the reported effectiveness of the drug in patients deficient in the LDL receptor. Initial studies of LDL fractional catabolic rate (FCR) using 125I-labeled LDL prepared from the serum of untreated WHHL rabbits showed no difference between probucol-treated WHHL rabbits and untreated WHHL rabbits. When, however, 125I-labeled LDL was prepared from donor WHHL rabbits under treatment with probucol and injected back into them, the FCR was found to be increased by about 50% above that measured simultaneously using 131I-labeled LDL prepared from untreated WHHL donors. The labeled LDL from probucol-treated donors was also metabolized more rapidly than that from untreated donors when injected into untreated WHHL rabbits or into untreated wild-type New Zealand White rabbits. Finally, it was shown that rabbit skin fibroblasts in culture degraded labeled LDL prepared from probucol-treated WHHL rabbits more rapidly than that prepared from untreated WHHL donors. This was true both for normal rabbit fibroblasts and also for WHHL skin fibroblasts, although the absolute degradation rates in the latter were, of course, much lower for both forms of LDL. The data indicate that a major mechanism by which probucol lowers LDL levels relates not to changes in the cellular mechanisms for LDL uptake or to changes in LDL production but rather to intrinsic changes in the structure and metabolism of the plasma LDL of the probucol-treated animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

PD123319 Augments Angiotensin II-Induced Abdominal Aortic Aneurysms through an AT2 Receptor-Independent Mechanism

Background

AT2 receptors have an unclear function on development of abdominal aortic aneurysms (AAAs), although a pharmacological approach using the AT2 receptor antagonist PD123319 has implicated a role. The purpose of the present study was to determine the role of AT2 receptors in AngII-induced AAAs using a combination of genetic and pharmacological approaches. We also defined effects of AT2 receptors in AngII-induced atherosclerosis and thoracic aortic aneurysms.

Methods and Results

Male AT2 receptor wild type (AT2 +/y) and deficient (AT2 -/y) mice in an LDL receptor −/− background were fed a saturated-fat enriched diet, and infused with either saline or AngII (500 ng/kg/min). AT2 receptor deficiency had no significant effect on systolic blood pressure during AngII-infusion. While AngII infusion induced AAAs, AT2 receptor deficiency did not significantly affect either maximal width of the suprarenal aorta or incidence of AAAs. The AT2 receptor antagonist PD123319 (3 mg/kg/day) and AngII were co-infused into male LDL receptor −/− mice that were either AT2 +/y or −/y. PD123319 had no significant effect on systolic blood pressure in either wild type or AT2 receptor deficient mice. Consistent with our previous findings, PD123319 increased AngII-induced AAAs. However, this effect of PD123319 occurred irrespective of AT2 receptor genotype. Neither AT2 receptor deficiency nor PD123319 had any significant effect on AngII-induced thoracic aortic aneurysms or atherosclerosis.

Conclusions

AT2 receptor deficiency does not affect AngII-induced AAAs, thoracic aortic aneurysms and atherosclerosis. PD123319 augments AngII-induced AAAs through an AT2 receptor-independent mechanism.     

Localization of lipoprotein in pre- and post-transition atherosclerotic lesions following short-term incubation with [125I]LDL

Summary Ultrastructural autoradiography has been used to test the hypothesis that atherosclerotic regions of vessels differ with respect to lipoprotein uptake and localization. White Carneau pigeons, in which the prevalence and localization of aortic lesions are highly predictable, were fed a 0.25% cholesterol-supplemented diet to accelerate atherosclerosis. One hour prior to necropsy the birds were given a single intravenous injection of homologous [¹²⁵I]LDL (low-density lipoprotein). Plasma die-away and tissue distribution of label were determined, and after the birds had been killed, the aortas, spleen and liver were processed for electron microscope autoradiography. Initial [¹²⁵I]LDL uptake was rapid, with 35% of the label removed within 30 min. Predominant accumulation was in the liver, followed by the lung, kidney, the spleen and the aorta, in which the [¹²⁵I]LDL level was approximately 4% that of the liver. Autoradiographic analysis documented hepatocyte (33%) and Kupffer cell (19.9%) localization in the liver and reticuloendothelial cell (57.4%) localization in the spleen. The aortic analysis involved serially sectioned lesions for direct comparison of non-lesion, lesion/non-lesion interface (edge) and deep lesion regions. Analysis of 2275 silver grains documented a ten-fold increase in LDL accumulation at the lesion edge (as compared to adjacent non-lesion) where macrophage foam cells contained more than 70% of the label. The other 30% was distributed equally among endothelium, the intimai matrix and smooth muscle cells. This distribution changed with more complex (deeper) lesions, although grain density in the complex lesions was comparable to the edge. In the complex regions, macrophage foam cell grains were reduced to 37%, whereas smooth muscle cell (22%) and the extracellular matrix (24%) label were both increased. These studies substantiate enhanced accumulation of lipoprotein specifically at lesion sites in the aorta and demonstrate a shift from macrophage localization at the developing edge to smooth muscle cell and the extracellular matrix in more complex deeper lesions.     

Localization of proteoheparan sulfate in rat aorta

Summary This study describes the distribution of heparan sulfate proteoglycan (HSPG) within the rat aorta using immunocytochemical (biotin-avidin-peroxidase) and immunoelectron microscopy (¹²⁵I-autoradiography). Heparan sulfate proteoglycan was isolated from a basement membrane producing mouse EHS sarcoma (Hassell et al. 1980) and used to generate antisera in rabbits. Light microscopic observations revealed intense immunostaining of the intima and media of normal aorta, adventitial vasa vasorum, and aortic intimal fibromuscular thickenings induced by experimental injury (balloon de-endothelialization). Immunoelectron microscopy using ¹²⁵I labeled antibodies to HSPG revealed that proteoheparan sulfate was localized to the amorphous layer of basement membrane below aortic and capillary endothelium. In addition, labeled anti-HSPG could be localized to the external lamina surrounding the smooth muscle cells in the hyperplastic intima. These studies reveal that antibodies prepared against a proteoheparan sulfate isolated from a basement membrane producting EHS sarcoma cross react with basement membrane structures within the aortic wall. Furthermore, these results demonstrate that the basement membranes beneath aortic and capillary endothelium and the external lamina surrounding aortic smooth muscle cells contain a heparan sulfate proteoglycan that is antigenically similar.     

Turnover of low density lipoproteins in normal and atherosclerotic rabbits

The turnover and composition of normal and hyperlipemic (h.l.) low density lipoproteins (LDL) of rabbits, were studied. They were obtained by ultracentrifugation and labeled by Bolton and Hunter method. Normal and h.l. LDL labeled with 125I were injected directly and crossed to both groups of rabbits. Normal and h.l. LDL had a different protein/lipid ratio. The analysis of fractional catabolic rate of LDL and the half-life of the phases of rapid and slow decay, show that h.l. LDL had a fractional catabolic rate that is the half of normal LDL and an increased half life of the phases of rapid and slow decay. Apparently, two factors: a) defective LDL receptor in the h.l. rabbit and b) different physico-chemical properties between normal and h.l. LDL, would be the reason for this difference. Besides, when normal and h.l. 125I LDL were injected into h.l. and normal rabbit, respectively, LDL changed according to the injected rabbit, as can be deduced from the analysis of the half life of the phase of slow decay.     

Fatty streak development in the vicinity of aortic coarctations in hypercholesterolemic rabbits

The influence of altered local hemodynamics on fatty streak development in rabbits fed high cholesterol diets was investigated. An aortic coarctation was created in the abdominal aorta of nine rabbits by placing a partially constricting gold or silver band (1.7 mm x 10 mm) around the aorta between the renal arteries and aortic bifurcation. Controls were 20 rabbits; seven sham operated and 13 unoperated rabbits. The abdominal aorta 1-2 cm proximal to the coarctation showed lipid deposition involving 45 +/- 8% (mean +/- SEM) of the luminal surface which was more than occurred within or distal to the obstruction (p less than 0.05) and also more than in controls (p less than 0.05). Within the coarctation, 4 +/- 2% of the luminal surface showed lipid deposition which was less than either proximally or distally (p less than 0.01) and also less than in comparable regions in controls (p less than 0.05). The aorta 1-2 cm distal to the coarctation showed lipid deposition involving 18 +/- 4% of the surface which was similar to control rabbits. Lipid deposition in corresponding regions of the control rabbits was involved in 17 +/- 4%, 19 +/- 5% and 19 +/- 4% of the luminal surface, respectively. Fatty streak development, therefore, appeared to be inhibited within the coarctation and enhanced proximal to it. The results suggest that some early step in the process of lipid accumulation may be affected by local fluid dynamics or modification of the wall of the vessel.     

Anti-atherogenic effect of coenzyme Q10 in apolipoprotein E gene knockout mice

Oxidation of low-density lipoprotein (LDL) lipid is implicated in atherogenesis and certain antioxidants inhibit atherosclerosis. Ubiquinol-10 (CoQ10H2) inhibits LDL lipid peroxidation in vitro although it is not known whether such activity occurs in vivo, and, if so, whether this is anti-atherogenic. We therefore tested the effect of ubiquinone-10 (CoQ10) supplemented at 1% (w/w) on aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice fed a high-fat diet. Hydroperoxides of cholesteryl esters and triacylglycerols (together referred to as LOOH) and their corresponding alcohols were used as the marker for lipoprotein lipid oxidation. Atherosclerosis was assessed by morphometry at the aortic root, proximal and distal arch, and the descending thoracic and abdominal aorta. Compared to controls, CoQ10-treatment increased plasma coenzyme Q, ascorbate, and the CoQ10H2:CoQ10 + CoQ10H2 ratio, decreased plasma alpha-tocopherol (alpha-TOH), and had no effect on cholesterol and cholesterylester alcohols (CE-OH). Plasma from CoQ10-supplemented mice was more resistant to ex vivo lipid peroxidation. CoQ10 treatment increased aortic coenzyme Q and alpha-TOH and decreased the absolute concentration of LOOH, whereas tissue cholesterol, cholesteryl esters, CE-OH, and LOOH expressed per bisallylic hydrogen-containing lipids were not significantly different. CoQ10-treatment significantly decreased lesion size in the aortic root and the ascending and the descending aorta. Together these data show that CoQ10 decreases the absolute concentration of aortic LOOH and atherosclerosis in apoE-/- mice.     

Residualizing and non-residualizing analogues of low-density lipoprotein as iodine-123 radiopharmaceuticals for imaging LDL catabolism

Low-density lipoprotein (LDL) labeled via direct iodination or via the radioiodinated residualizing moiety tyramine-cellobiose (TC) were compared in rabbits as potential ¹²³I radiopharmaceuticals for imaging sites of LDL catabolism. The tissue deposition of ¹³¹I-TC-LDL after 24 h as determined by dissection was in the major catabolic organs (liver, adrenals, spleen), and its plasma clearance was slower in rabbits with dietary hypercholesterolemia than in normals. ¹³¹I-LDL was unsuitable as a metabolic tracer due to redistribution of catabolites and/or loss of the label before protein degradation, which resulted in little accumulation of radioactivity in catabolic organs and high thyroid uptake. The plasma clearance half-time was similar (ca 22 h) for the two compounds in normal rabbits, but was increased to about 36 h for ¹³¹I-TC-LDL and decreased to approximately 9 h for ¹³¹I-LDL in hypercholesterolemic animals. The were similar with dynamic imaging of control and hypercholesterolemic rabbits using ¹²³I-labeled analogues. ¹²³I-TC-LDL rapidly localized in the liver, with low thyroid accumulation of radioactivity. The hepatic uptake of ¹²³I-LDL was about half that of ¹²³I-TC-LDL, and thyroid sequestration of radioactivity was significant for ¹²³I-LDL but not ¹²³I-TC-LDL. These data suggest that whereas the residualizing ¹²³I-TC-LDL has a pharmacokinetic profile representative of lipoprotein metabolism, the biodistribution of the activity from injected ¹²³I-LDL is complicated by processes other than protein degradation. The results are discussed with regard to nuclear medicine applications in evaluating lipoprotein catabolism in man.     

Evaluation of rat and rabbit sera lipoproteins in experimentally induced hyperlipidemia by analytical ultracentrifugation

Animals of various species are widely used as models with which to study atherosclerosis and the lipoprotein metabolism. The objective of this study was to investigate the lipoprotein profiles in Wistar rats and New Zealand white rabbits with experimentally induced hyperlipidemia by means of ultracentrifugation. The Schlieren curves were utilized to compare suckling and adult rat sera to determine whether aging causes alterations in lipoprotein profiles. A striking feature of the data is the high concentration of low-density lipoproteins (LDL), (>5.2 mmol/l cholesterol) in the 2-week old rat serum pool which was greatly decreased in the 3-weeks rat serum pool (<1.3 mmol/l cholesterol). Additional experiments were performed to permit a direct comparison of the amounts of lipoprotein present in rat sera in experimental hyperlipidemia post-Triton WR 1339 administration. Rapid changes in concentrations in very low-density lipoproteins (VLDL), LDL and high-density lipoproteins (HDL) were observed after Triton injection. The administration of Triton WR 1339 to fasted rats resulted in an elevation of serum cholesterol levels. Triton physically alters VLDL, rendering them refractive to the action of lipolytic enzymes in the blood and tissues, preventing or delaying their removal from the blood. Whereas the VLDL concentration was increased markedly, those of LDL and HDL were decreased at 20 h after Triton treatment. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate of LDL aliquots, to prepare radioactive-labeled lipoproteins and to study induced hyperlipidemia in rabbits. Analytical ultracentrifugation was applied to investigate the LDL flotation peaks before and after cholesterol feeding of rabbits. Modified forms of LDL were detected in the plasma of rabbits with experimentally induced atherosclerosis. ApoB-containing particles, migrating as LDL, intermediate density lipoproteins and VLDL were the most abundant lipoproteins. Gamma camera in vivo scintigraphy on rabbits with radiolabeled lipoproteins revealed visible signals corresponding to atherosclerotic plaques of the aorta and carotid arteries.