Progression of coronary atherosclerosis relates to the onset of myocardial infarction in an animal model of spontaneous myocardial infarction (WHHLMI rabbits).

Recently, we developed myocardial infarction-prone WHHLMI rabbits from coronary atherosclerosis-prone WHHL rabbits (WHHLCA rabbits) by selective breeding. In this study, we examined the relation of atherosclerotic plaques to the onset of myocardial infarction. We examined myocardial lesions of 378 WHHL rabbits born between 1992 and 2000, and atherosclerosis lesions of 93 WHHLCA and 82 WHHLMI rabbits. The aortic lesions were evaluated as percent surface lesion area. The coronary lesions were evaluated as cross sectional narrowing using sections prepared at 500 or 1,000 microm intervals. Serum lipid levels were assayed with enzymatic methods. The cumulative incidence of fatal myocardial infarction between 11 and 35 months old was 90% in WHHLMI rabbits and 21% in WHHLCA rabbits, respectively. Selective breeding increased the serum cholesterol levels by about 200 mg/dl despite there being no changes in triglyceride levels. Aortic and coronary atherosclerosis progressed markedly in WHHLMI rabbits compared to WHHLCA rabbits. Especially, WHHLMI rabbits over 15 months old showed more than 90% cross sectional narrowing of the left circumflex arteries, main stem of the left coronary artery, and the origin portion of the right coronary artery. In addition, there were no gender differences in atherosclerotic lesions of both aortas and coronary arteries. In conclusion, the present study showed that marked progression of coronary atherosclerosis was probably associated with spontaneous development of myocardial infarction in WHHLMI rabbits.     

Bisphenol A Exposure Enhances Atherosclerosis in WHHL Rabbits

Bisphenol A (BPA) is an environmental endocrine disrupter. Excess exposure to BPA may increase susceptibility to many metabolic disorders, but it is unclear whether BPA exposure has any adverse effects on the development of atherosclerosis. To determine whether there are such effects, we investigated the response of Watanabe heritable hyperlipidemic (WHHL) rabbits to 400-µg/kg BPA per day, administered orally by gavage, over the course of 12 weeks and compared aortic and coronary atherosclerosis in these rabbits to the vehicle group using histological and morphometric methods. In addition, serum BPA, cytokines levels and plasma lipids as well as pathologic changes in liver, adipose and heart were analyzed. Moreover, we treated human umbilical cord vein endothelial cells (HUVECs) and rabbit aortic smooth muscle cells (SMCs) with different doses of BPA to investigate the underlying molecular mechanisms involved in BPA action(s). BPA treatment did not change the plasma lipids and body weights of the WHHL rabbits; however, the gross atherosclerotic lesion area in the aortic arch was increased by 57% compared to the vehicle group. Histological and immunohistochemical analyses revealed marked increases in advanced lesions (37%) accompanied by smooth muscle cells (60%) but no significant changes in the numbers of macrophages. With regard to coronary atherosclerosis, incidents of coronary stenosis increased by 11% and smooth muscle cells increased by 73% compared to the vehicle group. Furthermore, BPA-treated WHHL rabbits showed increased adipose accumulation and hepatic and myocardial injuries accompanied by up-regulation of endoplasmic reticulum (ER) stress and inflammatory and lipid metabolism markers in livers. Treatment with BPA also induced the expression of ER stress and inflammation related genes in cultured HUVECs. These results demonstrate for the first time that BPA exposure may increase susceptibility to atherosclerosis in WHHL rabbits.     

Control of variance in experimental studies of hyperlipidemia using the WHHL rabbit

Between-animal variability has frustrated many experimental studies in outbred animal models of human disease. Variability that arises from genetic heterozygosity can be minimized by use of experimental designs that match littermates (polyzygotic twins) across control and treatment groups. Poor breeding vigor has prevented use of this experimental design in the WHHL rabbit model of hyperlipidemia and atherosclerosis. A comparison of reproduction in WHHL and normal rabbits demonstrated that litter size is limited by functional deficits at ovulation, implantation, and gestation in WHHL females. Superovulation of females reliably produced expanded litters of WHHL rabbits. Plasma lipids were measured in expanded litters of Japanese White WHHL (JW-WW) and English Half-lop WHHL (EHL-WW) rabbits. The variance of plasma cholesterol within sibships was two- to three-fold less than that between-litters. Intraclass correlation of total cholesterol within litters of EHL-WW was 0.72 and within litters of JW-WW was 0.67. These data provide evidence of genetic modulation of hypercholesterolemia in WHHL rabbits and demonstrate that experimental designs in which littermates are paired across groups can decrease the number of animals needed or increase the sensitivity of hypothesis tests by two- to threefold.     

Hypercholesterolemia inhibits L-type calcium current in coronary macro-, not microcirculation.

Hypercholesterolemia (HC) is a mary risk factor for the development of coronary heart disease. Coronary ion regulation, especially calcium, is thought to be important in coronary heart disease development; however, the influence of high dietary fat and cholesterol on coronary arterial smooth muscle (CASM) ion channels is unknown. The purpose of this study was to determine the effect of diet-induced HC on CASM voltage-gated calcium current (I(Ca)). Male miniature swine were fed a high-fat, high-cholesterol diet (40% kcal fat, 2% wt cholesterol) for 20-24 wk, resulting in elevated serum total and low-density lipoprotein cholesterol. Histochemistry indicated early atherosclerosis in large coronary arteries. CASM were isolated from the right coronary artery (>1.0 mm ID), small arteries ( approximately 200 microm), and large arterioles ( approximately 100 microm). I(Ca) was determined by whole cell voltage clamp. L-type I(Ca) was reduced approximately 30% by HC compared with controls in the right coronary artery (-5.29 +/- 0.42 vs. -7.59 +/- 0.41 pA/pF) but not the microcirculation (small artery, -8.39 +/- 0.80 vs. -10.13 +/- 0.60; arterioles, -10.78 +/- 0.93 vs. -11.31 +/- 0.95 pA/pF). Voltage-dependent activation was unaffected by HC in both the macro- and microcirculation. L-type voltage-gated calcium channel (Ca(v)1.2) mRNA and membrane protein levels were unaffected by HC. Inhibition of I(Ca) by HC was reversed in vitro by the cholesterol scavenger methyl-beta-cyclodextrin and mimicked in control CASM by incubation with the cholesterol donor cholesterol:methyl-beta-cyclodextrin. These data indicate that CASM L-type I(Ca) is decreased in large coronary arteries in early stages of atherosclerosis, whereas I(Ca) in the microcirculation is unaffected. The inhibition of calcium channel activity in CASM of large coronary arteries is likely due to increases in membrane free cholesterol.     

Lipoprotein(a) enhances advanced atherosclerosis and vascular calcification in WHHL transgenic rabbits expressing human apolipoprotein(a)

High lipoprotein(a) (Lp(a)) levels are a major risk factor for the development of atherosclerosis. The risk of elevated Lp(a) concentration is increased significantly in patients who also have high levels of low density lipoprotein (LDL) cholesterol. To test the hypothesis that increased plasma levels of Lp(a) may enhance the development of atherosclerosis in the setting of hypercholesterolemia, we generated Watanabe heritable hyperlipidemic (WHHL) transgenic (Tg) rabbits expressing human apolipoprotein(a) (apo(a)). We report here that Tg WHHL rabbits developed more extensive advanced atherosclerotic lesions than did non-Tg WHHL rabbits. In particular, the advanced atherosclerotic lesions in Tg WHHL rabbits were frequently associated with calcification, which was barely evident in non-Tg WHHL rabbits. To investigate the molecular mechanism of Lp(a)-induced vascular calcification, we examined the effect of human Lp(a) on cultured rabbit aortic smooth muscle cells and found that smooth muscle cells treated with Lp(a) showed increased alkaline phosphatase activity and enhanced calcium accumulation. These results demonstrate for the first time that Lp(a) accelerates advanced atherosclerotic lesion formation and may play an important role in vascular calcification.     

Hyaluronan accumulation is elevated in cultures of low density lipoprotein receptor-deficient cells and is altered by manipulation of cell cholesterol content

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2-4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2x10(6) Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.     

Arterial fatty acid-binding protein activity associated with dietarily-induced and spontaneously occurring atherosclerosis in the rabbit (Oryctolagus cuniculus)

1. Adult WHHL rabbits, or New Zealand rabbits fed either a stock chow diet or a high cholesterol diet were evaluated to assess the relationship between the development of aortic atherosclerosis and arterial FABP activity. 2. Aortic FABP activity was significantly (P less than 0.05) lower in atherosclerotic New Zealand aortas (0.039 +/- 0.008 nmol palmitoyl CoA bound/mg soluble prot) which had developed macroscopic lesions on 80% of the aortic surface as compared to lesion-free New Zealand aortas (0.053 +/- 0.002 nmol palmitoyl CoA bound/mg soluble prot). 3. In spontaneously hyperlipidemic rabbit (WHHL) aortas, FABP activity (0.023 +/- 0.004 nmol palmitoyl CoA bound/mg soluble prot) was significantly lower (P less than 0.05) than in either the normal or atherosclerotic New Zealand aortas. 4. To our knowledge, this study is the first to report a change in arterial FABP with the atherogenic process.     

Short-term therapy of atherosclerosis with low dose indomethacin: an experimental study

The effects of low dose indomethacin therapy in primary prevention of diet-induced atherosclerosis of rhesus monkeys was studied. The parameters studied were serum cholesterol concentration, thromboxane A2 (T x B2), prostacyclin (6-keto-PGF1 alpha) in serum/plasma, and the extent and intensity of coronary atherosclerosis. Although indomethacin did not affect serum cholesterol, it reduced serum T x B2 significantly (P less than 0.01). Plasma 6-keto-PGF1 alpha was not restored to the pretreatment level. A significant protective role of the drug was noted as far as coronary atherosclerosis is concerned (P less than 0.01).     

The influence of diet and carnitine supplementation on plasma carnitine, cholesterol and triglyceride in WHHL (Watanabe-heritable hyperlipidemic), Netherland dwarf and New Zealand rabbits (Oryctolagus cuniculus)

1. Plasma carnitine levels in the spontaneously (endogenously) hyperlipidemic Watanabe (WHHL) rabbit are approximately 2-fold higher (P less than 0.001) than in normal rabbits of the New Zealand (NZ) or Netherland Dwarf (NDw) breeds. 2. Plasma carnitine levels in WHHL (44 +/- 3 nmol/ml) can be approximated in NZ and NDw which are rendered exogenously hyperlipidemic by supplementation of the stock chow diet with cholesterol and peanut oil. 3. The induction of endogenous hyperlipidemia in NZ by feeding a sucrose casein rich diet results in a biphasic response of plasma carnitine (elevation followed by normalization). 4. Plasma carnitine in WHHL is readily elevated by supplemental L-carnitine and the elevation is associated with a reduction in plasma triglyceride which shows differences in individual response time; plasma cholesterol is unaffected by supplemental L-carnitine.     

Occurrence of sulfatide as a major glycosphingolipid in WHHL rabbit serum lipoproteins

Glycosphingolipids in serum and lipoproteins from Watanabe hereditable hyperlipidemic rabbit (WHHL rabbit), which is an animal model for human familial hypercholesterolemia (FH), were analyzed for the first time in this study. Chylomicrons and very low density, low density, and high density lipoproteins contained sulfatide as a major glycosphingolipid (12 nmol/mumol total phospholipids (PL) in chylomicrons, 19 nmol/mumol PL in VLDL, 18 nmol/mumol PL in LDL, and 14 nmol/mumol PL in HDL) with other minor glycosphingolipids such as glucosylceramide, galactosylceramide, GM3 ganglioside, lactosylceramide, and globotriaosylceramide. The concentration of sulfatide as a major glycosphingolipid in WHHL rabbit serum (121 nmol/ml) was much higher than that in normal rabbit serum (3 nmol/ml). Fatty acids of the sulfatides comprised mainly nonhydroxy fatty acids (C22, 23, and 24) and significant amounts of hydroxy fatty acids (about 10%) whereas long chain bases of the sulfatides comprised mostly (4E)-sphingenine with a significant amount of 4D-hydroxysphinganine (about 10%). Furthermore, sulfatides in the liver and small intestine from normal and WHHL rabbits (where serum lipoproteins are produced) were determined to amount to 260 nmol/g liver in WHHL rabbit, 104 nmol/g liver in control rabbit, 99.6 nmol/g small intestine in WHHL rabbit, and 31.2 nmol/g small intestine in control rabbit. Ceramide portions of the sulfatides in the liver were mainly composed of (4E)-sphingenine and nonhydroxy fatty acids, while those in the small intestine were mainly composed of 4D-hydroxysphinganine and hydroxy fatty acids. These results indicated that the sulfatides of serum lipoproteins were mostly derived from the liver (90% of the total), and that the remaining sulfatides (10% of the total) might be derived from the small intestine. These two sulfatides, which have different ceramide portions, could be useful markers for metabolic and biosynthetic studies of various lipoproteins in WHHL rabbit, and thus would be helpful to further elucidate the relationship between hypercholesterolemia and atherosclerosis in the rabbit.     

The effect of pravastatin on serum cholesterol levels in hypercholesterolemic diabetic rabbits

Diabetes mellitus is associated with hyperlipidemia and increased risk of atherosclerosis. A diabetic animal model has been developed to study the effect of treatment with pravastatin, a potent HMG CoA reductase inhibitor, on plasma lipoprotein levels. Hypercholesterolemia was induced in alloxan diabetic and control rabbits by feeding a diet containing 25% casein and 10% hydrogenated coconut oil for 8 weeks. Feeding the casein-coconut oil diet to the diabetic group resulted in a 5-fold increase in serum cholesterol levels, which was not statistically different from the nondiabetic group fed this diet. However, in the diabetic group, there was more cholesterol in the VLDL fraction and less in LDL as compared to the nondiabetic group. Serum triacylglycerol levels in the diabetic rabbits were variable and ranged from 58-943 mg/dl. The diabetic and nondiabetic animals were then treated with pravastatin at a dose of 10 mg/kg per day for 21 days. In the nondiabetic group, pravastatin treatment significantly lowered serum and LDL cholesterol concentrations by 28.5% (52.3 mg/dl, P less than 0.05) and 36.2% (40.7 mg/dl, P less than 0.05) respectively, relative to the placebo group. Serum and VLDL triacylglycerol levels in the nondiabetic group were also significantly decreased following pravastatin treatment. In the diabetic group, serum and LDL cholesterol levels were decreased by 37.0% (69.1 mg/dl, P less than 0.05) and 52.7% (32.1 mg/dl, P less than 0.01), respectively, relative to the diabetics given the placebo. Pravastatin treatment did not adversely affect serum glucose levels. Thus, pravastatin treatment was effective in controlling the hypercholesterolemia present in these diabetic animals.     

Transgenic rabbits as models for atherosclerosis research

Several characteristics of the rabbit make it an excellent model for the study of lipoprotein metabolism and atherosclerosis. New Zealand White (NZW) rabbits have low plasma total cholesterol concentrations, high cholesteryl ester transfer protein activity, low hepatic lipase (HL) activity, and lack an analogue of human apolipoprotein (apo) A-II, providing a unique system in which to assess the effects of human transgenes on plasma lipoproteins and atherosclerosis susceptibility. Additionally, rabbit models of human lipoprotein disorders, such as the Watanabe Heritable Hyperlipidemic (WHHL) and St. Thomas' Hospital strains, models of familial hypercholesterolemia and familial combined hyperlipidemia, respectively, allow for the assessment of candidate genes for potential use in the treatment of dyslipoproteinemic patients. To date, transgenes for human apo(a), apoA-I, apoB, apoE2, apoE3, HL, and lecithin:cholesterol acyltransferase (LCAT), as well as for rabbit apolipoprotein B mRNA-editing enzyme catalytic poly-peptide 1 (APOBEC-1), have been expressed in NZW rabbits, whereas only those for human apoA-I and LCAT have been introduced into the WHHL background. All of these transgenes have been shown to have significant effects on plasma lipoprotein concentrations. In both NZW and WHHL rabbits, human apoA-I expression was associated with a significant reduction in the extent of aortic atherosclerosis, which was similarly the case for LCAT in rabbits having at least one functional LDL receptor allele. Conversely, expression of apoE2 in NZW rabbits caused increased susceptibility to atherosclerosis. These studies provide new insights into the mechanisms responsible for the development of atherosclerosis, emphasizing the strength of the rabbit model in cardiovascular disease research.     

Role of receptor-independent low density lipoprotein transport in the maintenance of tissue cholesterol balance in the normal and WHHL rabbit

These studies were undertaken to determine the role of receptor-independent low density lipoprotein (LDL) transport in cholesterol balance across individual tissues and the whole animal. Homologous LDL, which measures total LDL transport, and methylated heterologous LDL, which measures receptor-independent LDL uptake, were cleared from the plasma at very different rates in the NZ control rabbit (3,900 and 1,010 microliter/hr per kg, respectively) whereas in the WHHL rabbit both preparations were cleared at essentially the same rate (approximately 1,070 microliter/hr per kg). Receptor-independent LDL clearance was detected in all tissues of the NZ control rabbit and these varied from 32 (spleen) to less than 0.5 (skeletal muscle) microliter/hr per g. In contrast, receptor-dependent LDL uptake was found in only about half of these same organs. In the WHHL rabbit, the rates of receptor-independent LDL transport were the same as in the NZ control rabbit, but no receptor-dependent uptake was detected. Using these clearance values it was calculated that in the control rabbit nearly 70% of LDL-cholesterol was removed from the plasma by the liver and 89% of this was receptor-mediated. With loss of receptor activity, however, the burden of LDL degradation was shifted away from the liver so that approximately 70% of LDL-cholesterol uptake took place in the extra-hepatic tissues of the WHHL rabbit. Thus, in the normal animal, the primary function of receptor-dependent LDL transport is to promote the rapid uptake and disposal of plasma LDL by the liver. In the absence of such receptor activity, cholesterol balance across most individual organs and the whole animal remains essentially normal and is mediated by the receptor-independent process. Because of the much lower absolute clearance rates manifested by this transport mechanism, however, substantial and predictable elevations in the circulating plasma LDL-cholesterol levels are required to maintain this balance.     

Characterization of very low density lipoprotein from Watanabe heritable hyperlipidemic rabbits

We investigated the properties of very low density lipoprotein (VLDL) from two types of Watanabe heritable hyperlipidemic (WHHL) rabbits: one with a high incidence of coronary atherosclerosis (type 1), and the other with a low incidence (type 2). When incubated with mouse peritoneal macrophages, VLDL from type 1 WHHL rabbit (type 1-VLDL) stimulated cholesteryl ester synthesis 10.5-fold more than VLDL from the type 2 WHHL rabbit (type 2-VLDL) did. Moreover, a similar difference was seen in the stimulation of cholesteryl ester synthesis in peritoneal macrophages isolated from the WHHL rabbits. The mass ratios of cholesterol to protein in type 1- and type 2-VLDL were 5.69 and 2.05, respectively. Agarose gel electrophoresis of type 1-VLDL showed beta mobility, and that of type 2-VLDL showed pre-beta mobility. No difference was seen between the sizes of VLDL particles of the two types. The amount of apolipoprotein E in type 1-VLDL was greater than that in type 2-VLDL. In conclusion, the difference between type 1 and type 2 WHHL rabbits is at least partly due to the presence in type 1 animals of VLDL particles rich in cholesteryl esters and apolipoprotein E, particles which are very similar to beta-VLDL in conformation.     

G-CSF prevents the progression of atherosclerosis and neointimal formation in rabbits

Granulocyte colony-stimulating factor (G-CSF) prevents left ventricular remodeling after myocardial infarction, but its effect on atherosclerosis is unknown. We examined two kinds of rabbit atherosclerosis models. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHL-MI) rabbits were treated with G-CSF or saline for 7 days from 14 months old. The vascular injury models were created by inflating angioplasty balloon in the iliac artery of rabbits and were divided into G-CSF and saline group. G-CSF significantly reduced the stenosis score of coronary artery and lipid plaque area of thoracic aorta in WHHL-MI rabbits at 4 weeks after the treatment. In the vascular injury model, G-CSF significantly prevented an increase in neointima/media ratio at 4 weeks after the treatment. G-CSF accelerated the reendothelialization of denuded arteries, and the pretreatment with nitric oxide synthase inhibitor significantly inhibited it. These results suggest that G-CSF has a therapeutic potential for the progression of atherosclerosis.     

Antibody against cholesteryl ester transfer protein (CETP) elicited by a recombinant chimeric enzyme vaccine attenuated atherosclerosis in a rabbit model

The recombinant chimeric enzyme of AnsB-TTP-CETPC, which comprised asparagianse, tetanus toxin helper T-cell epitope and CETP B-cell epitope, was used to vaccinate New Zealand white rabbits in alum adjuvant. After anti-CETP antibodies were successfully produced, rabbits were fed with a high cholesterol diet for fifteen weeks until atherosclerotic lesions formed in arteries. The results showed that after CETP was inhibited by anti-CETP antibodies the fraction of plasma cholesterol in HDL increased and the fraction of plasma cholesterol in LDL decreased in rAnsB-TTP-CETPC immunized rabbits. The average size of aorta atherosclerotic plaques in rabbits treated with rAnsB-TTP-CETPC was 42.3% less than in rabbits treated with OVA (neutral control), or 47.6% less than in rabbits treated with rHSP 65 (negative control). The average thickness of hyperplastic coronary artery in rAnsB-TTP-CETPC immunized rabbits was 159+/-12 microm, which was significantly lower than in rHSP 65 immunized rabbits (187+/-15 microm) or OVA immunized rabbits (248+/-18 microm). The data reported here demonstrated that rAnsB-TTP-CETPC could significantly attenuate the development of atherosclerosis in rabbits fed with high cholesterol diet, and might be developed to an anti-atherosclerosis vaccine in the future.     

小檗碱对动脉粥样硬化形成的抑制作用及机制探讨

目的:研究小檗碱(Berberine)对家兔动脉粥样硬化形成的抑制,并探讨其可能的作用机制。方法:将32只雄性新西兰大白兔随机分为正常组、模型对照组、小檗碱组和阿托伐他汀组,每组各8只。正常组以普通饲料喂养,其余各组高脂喂养,小檗碱组和阿托伐他汀组分别灌胃给予小檗碱(100 mg/kg,1次/d)和阿托伐他汀(5 mg/kg,1次/d),饲养12周。利用全自动生化分析仪测定兔血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C)水平,酶联免疫吸附法测定血清ox-LDL、MCP-1、MMP-9水平。观察斑块破裂和血栓形成情况,并进行苏木素-伊红(HE)染色,测量病变区域内膜与中膜的厚度。结果:小檗碱可显著降低高脂喂养家兔的血清TC、TG、LD-C水平,降低血清血清炎症因子ox-LDL、MCP-1、MMP-9水平(P0.01),与阿托伐他汀组相比无显著性差异。同时小檗碱组的内膜增生程度明显小于模型对照组,和阿托伐他汀组接近。结论:小檗碱可改善动脉粥样硬化病变的程度,抑制斑块的形成,同时降低血清中炎症因子标志物。     

Characterization of arachidonic acid metabolism in Watanabe heritable hyperlipidemic (WHHL) and New Zealand white (NZW) rabbit aortas

WHHL rabbits develop progressive atherosclerosis. There are no visible signs of the disease at 1 month, however, by 12 months, the formation of aortic plaques is extensive. This study characterized arachidonic acid (AA) metabolism in 1 and 12 month old WHHL and NZW rabbit aortas. Vessels incubated with 14C-AA and A23187 metabolized AA to a number of oxygenated products as identified by high pressure liquid chromatography. The major AA metabolites produced by WHHL and NZW aortas were 6-keto PGF1 alpha, PGE2, 12- and 15-hydroxyeicosatetraenoic acids (HETEs). The structures of the HETEs were confirmed by gas chromatography-mass spectrometry. Indomethacin blocked the synthesis of prostaglandins (PGs) but not HETEs whereas ETYA, NDGA or removal of the endothelium attenuated the production of both PGs and HETEs. Measurement of 6-keto PGF1 alpha, 12- and 15-HETE by specific radioimmunoassays indicated that as the rabbits aged and as atherosclerosis progressed, aortas lost the ability to synthesize 6-keto PGF1 alpha and 15-HETE. Prior to the development of atherosclerosis, 1 month old WHHL aortas produced 70% less 15-HETE than did NZW aortas. Atherosclerotic aortas from 12 month old WHHLs synthesized 60% less 6-keto PGF1 alpha during stimulation with AA or A23187 than did 12 month old NZW aortas. We conclude that the development and expression of atherosclerosis in WHHL rabbits impairs the ability of aortas to metabolize AA to both PGs and HETEs.     

Estimation of 26-hydroxycholesterol in serum by high-performance liquid chromatography and its measurement in patients with atherosclerosis

A method for analysing 26-hydroxycholesterol (26OHC) in serum and tissue samples using solid phase extraction and high-performance liquid chromatography is reported. This procedure was used to measure the levels of 26OHC in the sera of apparently healthy subjects and of 18 patients with angiographically proven atherosclerosis. Sixteen of the patients had levels within or below the range detected in the apparently healthy subjects (125-294 ng/ml), indicating that high 26OHC levels cannot be a major factor in the development of atherosclerosis. However, when the patients and the normal subjects were combined in a group, there was a significant positive correlation (r = 0.54, P less than 0.01) between serum cholesterol and serum 26OHC, and that correlation approached significance for each of the individual groups (P = 0.06 for each group). These results suggest that there is an association between cholesterol and 26OHC levels in human serum.     

Growth-related oncogene-alpha induces endothelial dysfunction through oxidative stress and downregulation of eNOS in porcine coronary arteries

Growth-related oncogene-alpha (GRO-alpha) is a member of the CXC chemokine family, which is involved in the inflammatory process including atherosclerosis. We hypothesized that GRO-alpha may affect endothelial functions in both porcine coronary arteries and human coronary artery endothelial cells (HCAECs). Vasomotor function was analyzed in response to thromboxane A2 analog U-46619 for contraction, bradykinin for endothelium-dependent vasorelaxation, and sodium nitroprusside (SNP) for endothelium-independent vasorelaxation. In response to 10(-6) M bradykinin, GRO-alpha (50 and 100 ng/ml) significantly reduced endothelium-dependent vasorelaxation by 34.73 and 48.8%, respectively, compared with controls (P < 0.05). There were no changes in response to U-46619 or SNP between treated and control groups. With the lucigenin-enhanced chemiluminescence assay, superoxide anion production in GRO-alpha-treated vessels (50 and 100 ng/ml) was significantly increased by 50 and 86%, respectively, compared with controls (P < 0.05). With real-time PCR analysis, endothelial nitric oxide synthase (eNOS) mRNA levels in porcine coronary arteries and HCAECs after GRO-alpha treatment were significantly decreased compared with controls (P < 0.05). The eNOS protein levels by both immunohistochemistry and Western blot analyses were also decreased in GRO-alpha-treated vessels. Antioxidant seleno-l-methionine and anti-GRO-alpha antibody effectively blocked these effects of GRO-alpha on both porcine coronary arteries and HCAECs. In addition, GRO-alpha immunoreactivity was substantially increased in the atherosclerotic regions compared with nonatherosclerotic regions in human coronary arteries. Thus GRO-alpha impairs endothelium-dependent vasorelaxation in porcine coronary arteries through a mechanism of overproduction of superoxide anion and downregulation of eNOS. GRO-alpha may contribute to human coronary artery disease.