Atherosclerosis

Published by Elsevier
Online ISSN: 0021-9150
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Article
The aim of this study was to evaluate the cholesterol-lowering and antiatherosclerotic effect of the HMG-CoA reductase inhibitor pravastatin sodium at a dosage comparable to human therapy. Twelve heterozygous WHHL rabbits (13 months old) were fed 100 g per day of a low cholesterol (0.03%) enriched diet for 12 months. Six of these animals also received pravastatin sodium at a daily dose of 1 mg/kg body weight (verum group). In the verum group, total plasma cholesterol levels were lower by 47%(P < 0.05) and relative aortic plaque volume (% ratio of total plaque volume to the aortic lumen) was reduced by 78% (P < 0.05), when compared to the control group. Plaque composition was analysed at 30 cross-sectional levels of the entire aortic wall using a grid window. Compared to the control group, the plaque type, in terms of architecture and composition, was altered as follows: lesions in the verum group had no confluent atheromatous cores and showed a pattern of a diffuse mixture of the main plaque components with a decreased relative content of necrosis (-44%) and an increased relative content of smooth muscle cells (+19%), whereas the relative content of macrophage-derived foam cells and collagen were nearly unaffected. Furthermore, a similar plaque volume and type was observed in animals with comparable cholesterol profiles. There was no histologic evidence for structurally damaging effects of pravastatin sodium on the arterial wall. We conclude that pravastatin sodium reduces total plasma cholesterol levels in this animal model, thereby leading to smaller plaques and a different plaque type.
 
Article
The effect of the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor HL-004 on bile acid production was studied during the regression phase of pre-established hypercholesterolemia in stroke-prone spontaneously hypertensive rats (SHRSP). These rats were fed a hypercholesterolemic diet containing 5% cholesterol, 2% cholic acid, and 20% suet for 30 days to induce hypercholesterolemia. The regression phase was started by switching the diet to normal chow, followed by another 30 days of the diet. The decrease in serum cholesterol level was accelerated by treatment with 0.09% HL-004. At the end of regression, hepatic ACAT activity was significantly lower in the HL-004 treated animals, an event concomitant with the significant decrease in cholesteryl ester content in the liver. In contrast hepatic cholesterol 7 alpha-hydroxylase activity was maintained at a higher level in the HL-004 treated animals. HL-004 increased the secretion of bile acid and biliary lipids in bile duct-cannulated SHRSP. In HepG2:cells, HL-004 at 1-30 microM dose-dependently stimulated bile acid synthesis from [3H]cholesterol. When cholesterol 7 alpha-hydroxylase activity of the liver was compared ex vivo in the presence and in the absence of exogenous cholesterol, it was suggested that the higher 7 alpha-hydroxylase activity of the HL-004 group could be attributed not only to expansion of the endogenous cholesterol pool, which may be the result of hepatic ACAT inhibition by HL-004 but to the direct effect of HL-004 on bile acid production. Thus, HL-004 accelerates the regression of hypercholesterolemia, an event which may be related to the stimulation of bile acid production in the liver.
 
Article
Molecules which egress from atherosclerotic arteries may function as plasma markers of arterial pathology, but such egress has not been proven with living human coronary arteries. We hypothesised that proteins eluting from the arterial wall may discriminate between atherosclerotic and non-atherosclerotic coronary arteries. During cardiac bypass surgery, 155 sequential fractions of antegradely flushed coronary cardioplegia solution were collected by balloon-cuffed catheter from the coronary sinus in subjects with angiographically extensive (n=30) or minor (n=7) coronary disease. Although plasma was the major source of protein in heavily blood-contaminated samples, under conditions of low blood contamination (<0.5 mg/ml red cell Haemoglobin) coronary circulation-derived protein was detected. N-terminal sequencing of a major 40 kDa band detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated 100% homology with beta chain of Haptoglobin (Hpt). Comparison of perfusates from patients with and without significant coronary disease found that the concentration of Hpt was markedly increased in perfusates from atherosclerotic coronary arteries (0.099+/-0.017 microg Hpt/microg Hb) relative to controls (0.016+/-0.008 microg Hpt/microg Hb, P=0.0027). Analysis of peripheral plasma samples of the same subjects, and of a separate cohort of patients, confirmed greater Hpt in those with angiographic coronary disease than in those without disease. Proteins such as Hpt elute from the human coronary vascular bed and may differentiate between arteries with minor or extensive atherosclerosis. Although the suitability of Hpt as a circulating plasma marker for atherosclerosis remains to be established, the approach used in the present study may permit identification of diverse plasma-detectable markers of atherosclerosis, and the subsequent non-invasive evaluation of in vivo arterial pathology.
 
Article
Major histocompatibility complex (MHC) gene region harbours haplotypes that associate with coronary artery disease (CAD). Their role in ST-elevation infarction (STEMI) or on the inflammatory level is not known. Four candidate MHC markers were analyzed by real-time quantitative PCR and constructed into haplotypes from patients with STEMI (n = 162), matched controls with no CAD (n = 319) and general population sample (n = 149). High sensitivity C-reactive protein (hsCRP) was assessed in a follow-up visit from patients (n = 86) and at inclusion from other study subjects. The haplotype with one copy of HLA-DRB1*01, C4A, C4B but no HLA-B*35 doubled the risk of STEMI (OR = 2.15, 95%CI = 1.11-4.15, p = 0.020 for patients vs. controls, and OR = 2.26, 95%CI = 0.97-5.24, p = 0.052 for patients vs. population sample). The association between patients and controls persisted in multivariate analyses. The frequency of the haplotype was 5.86% (n = 19/324) in patients, 2.82% (n = 18/638) in controls and 2.68% (n = 8/298) in population sample. None of the individual MHC markers alone showed significant association with STEMI. In multivariate analyses, the haplotype carriers had higher hsCRP levels in patients (median 3.37 mg/L in carriers vs. 1.14 mg/L in non-carriers, p = 0.019) and in controls (median 2.90 mg/L vs. 1.21 mg/L, p = 0.009, respectively). The MHC haplotype associates with STEMI and elevated baseline hsCRP levels. The results are in concordance with previous data on non-STEMI patients, implying that a HLA-DRB1*01--related haplotype increases the risk of CAD, possibly though increased inflammation.
 
Article
Ro 16-0521 is a newly synthesized benzodiazepine derivative which is devoid of reactivity with the brain benzodiazepine receptor. The effects of this drug on plasma lipids and lipoproteins and hepatic cholesterol metabolism have been examined in the cholesterol-fed rat. Drug therapy was associated with dose-related falls in plasma cholesterol concentration, liver cholesterol content, and the activity of the liver enzyme Acyl-CoA cholesterol acyltransferase. Drug therapy abolished the lipid and lipoprotein changes induced by cholesterol feeding, including those associated with a diet supplemented with olive oil to facilitate cholesterol loading. Drug therapy was also associated with an increased activity of the enzyme HMG-CoA reductase and reduced hepatic microsomal cholesterol content. It is suggested that the cholesterol-fed rat will be a suitable model for further mechanistic studies.
 
Article
Familial combined hyperlipidemia (FCHL) is characterized by elevated levels of serum total cholesterol (TC), triglyceride (TG), or both. The increased incidence of coronary artery diseases (CAD) in the patients with FCHL is believed to be caused by circulating atherogenic lipoproteins associated with the complex phenotype. Recent establishment of sensitive detection system for malondialdehyde-modified (MDA)-LDL, which is one of oxidized lipoproteins, showed its increased circulating level in the patients with CAD. In order to know the atherogenic lipoproteins resulted from the dyslipidemia observed in FCHL, we measured the serum MDA-LDL level in the patients. The circulating MDA-LDL level and the ratio of MDA-LDL and LDL-C in FCHL were significantly higher (P<0.05) than those in control, which are adjusted about the age, serum TC, LDL-C and HDL-C levels, respectively. Furthermore, the circulating MDA-LDL level and the ratio of MDA-LDL and LDL-C were negatively correlated (R=-0.635, P<0.01 and R=-0.702, P<0.01, respectively) with hepatic lipase (HL) activity in FCHL. The serum MDA-LDL level and the ratio of MDA-LDL and LDL-C were in the subjects with T/T genotypes in the HL C-514T polymorphism were significantly increased compared to those with C/C genotype, respectively. The subjects with T/T genotype showed the activities to 65 and 79% of those in the subjects with C/C genotype in male and female, respectively. The intima-media thickness (IMT) of carotid artery was significantly higher (P<0.05) in the subjects with T/T genotype than those with C/C genotype in male. These findings indicate that the circulating MDA-LDL level is possibly contributing the atherogenic process in FCHL, and the common HL polymorphism might be a determinant of the serum level of oxidized LDL in the patients with FCHL.
 
Article
Unlabelled: Oxidative stress and inflammation are key promoters of atherosclerosis and myocardial damage. When orally administered, the novel astaxanthin prodrug CDX-085 delivers high levels of the xanthophyll antioxidant astaxanthin that protects LDL from oxidation and reduces primary thrombosis. In this study, we analyzed whether delivery of astaxanthin from administration of the CDX-085 prodrug reduces plasma lipoprotein levels and the progression of atherosclerosis in low-density lipoprotein receptor negative (LDLR(-/-)) and apolipoprotein E deficient (ApoE(-/-)) mice. Methods: Relative circulating levels of astaxanthin derived from CDX-085 administration compared to administration of pure astaxanthin was initially evaluated in a canine model. In mouse Study #1, 16 wild-type and 16 LDLR(-/-) mice on 0.5% cholesterol diet supplemented with either 0.0%, 0.08%, 0.2% and 0.4% CDX-085 were used to assess plasma levels and lipoprotein biodistribution measured by FPLC after 4 weeks treatment. In Study #2, 36 male LDLR(-/-) mice were randomized to a 0.5% cholesterol chow diet (CHOW group, n=12) or 0.5% cholesterol chow fortified with 0.08% CDX-085 (n=12) or 0.5% cholesterol chow with 0.4% CDX-085 (n=12) for 12 weeks. In Study #3, 34 male ApoE(-/-) mice were randomized in the same fashion as the Study #2 and fed similar diets for 9 weeks. Results: CDX-085 administration was shown to result in significantly higher levels of circulating astaxanthin (p<0.001 ANOVA) over a 72 h period compared to pure, non-esterified astaxanthin in a single-dose pharmacokinetic study in beagles. In Study #1, plasma astaxanthin levels were 5-9-fold higher in LDLR(-/-) mice compared to wild-type mice. Astaxanthin was highly distributed among all lipoprotein fractions, generally reflecting cholesterol content of lipoproteins. In Study #2, administration of CDX-085 resulted in significantly lower total cholesterol levels (528±68 mg/dL vs. 550±67 mg/dL vs. 602±80 mg/dL, p=0.047) and aortic arch atherosclerosis (9.0±4.2% vs. 9.8±3.5% vs. 13.2±3.6%, p=0.023) in the 0.4% CDX-085 group compared to the 0.08% CDX-085 and CHOW groups, respectively. In ApoE(-/-) mice, a 72% reduction in triglycerides in the 0.4% CDX-085 group and 50% reduction in the 0.08% CDX-085 groups was noted compared to CHOW group (final levels 17±11 mg/dL vs. 30±15 mg/dL vs. 60±32 mg/dL, respectively, p=0.001). Conclusion: Oral administration of the novel astaxanthin prodrug CDX-085 shows that it distributes among lipoproteins. CDX-085 lowers total cholesterol and aortic arch atherosclerosis in LDLR(-/-) mice and triglyceride levels in ApoE(-/-) mice and shows promise for further evaluation in human studies.
 
Article
Administration of ethane-1-hydroxy-1,1-diphosphonate (EHDP) to swine with pre-established atherosclerosis resulted in lower lesion calcium concentration, smaller lesions and a decrease in the area of lesions involved in necrosis. Atherosclerosis was developed in Yorkshire swine by balloon catheter-injury to the abdominal aorta, followed by a high cholesterol-high lipid (HL) diet for 4 months. The administration of EHDP (20 mg/kg/day) was begun after these 4 months and continued for 5 additional months along with the atherogenic diet. Other swine were ballooned and fed HL diet for nine months. Morphometric analysis showed that the extent of lesions, expressed as ratio of intima to media was significantly less (P less than 0.05) in the EHDP-treated HL swine, compared to the HL diet-only group. The ratio of lesion areas showing lipid-rich necrotic debris to the area of media was also significantly smaller (P less than 0.05). Biochemical analysis showed that the lesion from the HL drug-treated group contained significantly less (P less than 0.05) calcium compared to that from the HL diet only. Finally, there was significant correlation between average lesion area and average lesion calcium concentration (P less than 0.02) for both groups. While the effect of EHDP on lesion size and calcium concentration has been previously reported for various species such as rabbit and monkey, this study is believed to be the first where a beneficial effect of EHDP on one of the most serious complications of atherogenesis - necrosis - has been documented. The mechanisms by which EHDP may affect necrosis are discussed.
 
Article
Atherosclerosis was induced in White Carneau pigeons by feeding cholesterol either continuously or intermittently during a 14-month period. Animals were then treated daily with either saline, or 0.5 or 2.5 mg EHDP/kg body wt during which time a cholesterol-free “regression” diet was fed. Subgroups of pigeons were studied after four and eight months of treatment. After the progression period, aortas and brachiocephalic arteries of those pigeons that were subjected to intermittent hypercholesterolemia accumulated more cholesterol, were more complicated, and showed a different pattern of lesion regression than arteries in animals continuously hypercholesterolemic. EHDP treatment had no effect on plasma cholesterol, triglyceride, calcium, or phosphorus concentrations. In animals with atherosclerosis induced by intermittent hypercholesterolemia, EHDP treatment resulted in a reduction in intimal plaques in the aorta and brachiocephalic arteries while in animals with atherosclerosis induced by continuous hypercholesterolemia EHDP treatment resulted in more extensive intimal plaques. In animals fed cholesterol intermittently and treated with EHDP, aortic cholesterol concentrations were lower; but animals with atherosclerosis induced by continuous cholesterol feeding and treated with EHDP had higher aortic cholesterol concentrations than controls. Among pigeons with atherosclerosis augmented by intermittent cholesterol feeding, there was increased mineralization during regression and this complication was found to be prevented by EHDP treatment. No effect was seen on collagen concentrations in either the intermittent or the continuous cholesterol fed animals. The beneficial effect of EHDP on the more advanced atherosclerosis in the pigeons fed cholesterol intermittently may be due to an action on lipid-calcium-connective tissue complexes within the atherosclerotic plaque. The opposite effect of EHDP in pigeons with relatively uncomplicated atherosclerosis induced by continuous cholesterol feeding is less easily understood, but a possible role implicating lysosomes is suggested.
 
Article
The purpose of this study was to test the effectiveness of various doses of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) in an experimental rabbit model of athero-arteriosclerosis designed by Hass et al. (Amer. J. Pathol., 49 (1966) 739). This model, which involves the feeding of a hypercholesterolemic diet in conjunction with the administration of moderately high doses of vitamin D and nicotine, results in an extensive arterial disease with complicated lesions. EHDP was administered daily by subcutaneous injection at levels of 0.25, 1.0 and 2.5 mg/kg body weight beginning with the initiation of the atherogenic regimen. Results of chemical and histopathological analyses after 8 and 12 weeks of treatment indicate the following: (1) There was a dose-related inhibition of arterial calcification at 8 weeks. At 12 weeks, only the 2.5 mg/kg dose of EHDP resulted in reduced calcification. (2) EHDP administration appeared to influence arterial lipid-containing plaque formation in medium sized arteries at 12 weeks. There was no apparent effect of EHDP administration on serum cholesterol and triglyceride levels. (3) EHDP, at a dose of 2.5 mg/kg/day, inhibited the vitamin D induced hypercalcemia. (4) EHDP administration at 2.5 mg/kg/day almost totally inhibited the thromboarteritis accompanying this disease. (5) The data thus indicate that if arterial calcification is inhibited, the other morphological effects of this treatment regime are also inhibited. This effect occurred even though serum lipid levels were unaffected. The data therefore emphasize the role of calcification in the pathogenesis of this type of experimental atherosclerosis and perhaps in human disease as well.
 
Article
There is increasing evidence that aldehydes, including acrolein generated endogenously during the degradation process of biological molecules or the metabolism of foreign chemicals may be involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis. Because glutathione (GSH) and GSH S-transferase (GST) are a major cellular defense against the toxic effects of reactive aldehydes, in this study we have characterized the inducibility of GSH and GST by the unique chemoprotective agent, 3H-1,2-dithiole-3-thione (D3T) and their protective effects against acrolein-induced toxicity in rat aortic smooth muscle A10 cells. Incubation of rat aortic A10 cells with micromolar concentrations of D3T resulted in a concentration- and time-dependent induction of both GSH and GST. Treatment of A10 cells with D3T also led to induction of gamma-glutamylcysteine synthetase, the key enzyme involved in GSH biosynthesis. Notably, the levels of GSH and GST remained higher than basal levels 72 h after removal of D3T from the culture media. To examine the protective effects of D3T-induced GSH and GST against reactive aldehyde-mediated toxicity, A10 cells were pretreated with D3T and then exposed to acrolein. Pretreatment of A10 cells with D3T resulted in a marked decrease of acrolein-induced toxicity as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay and morphological changes. To further demonstrate the involvement of GSH and GST in protecting against acrolein-induced toxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively. Either depletion of cellular GSH by BSO or inhibition of cellular GST by sulfasalazine led to a marked potentiation of acrolein-induced toxicity in A10 cells. Furthermore, co-treatment of cells with BSO was found to greatly abolish the protective effects of D3T on acrolein-induced toxicity. Taken together, our results demonstrate for the first time that both GSH and GST in aortic smooth muscle cells can be induced by D3T, and that this increased cellular defense affords great protection against reactive aldehyde-induced cardiovascular cell injury.
 
Article
The properties of CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT) (EC 2.7.8.2.), which catalyzes de novo synthesis of phosphatidylcholine, were studied in rat arterial wall. The optimal pH of CPT of the arterial wall was about 8.5. On subcellular fractionation of the arterial wall, the highest activity was found in the microsome-rich fraction; the cytosolic fraction showed only a trace of activity. The Michaelis constant (KM) for CDP-choline was 0.019 mM. The CPT activity of a homogenate of arterial wall increased linearly with increase in concentration of diolein up to 3.2 mM. 20 mM magnesium and 0.2 mM manganese ions caused marked activation respectively and essential for the activity. Calcium, barium, cobalt, copper, and ferrous ions were inhibitory. 0.5 mM ethylenediaminetetraacetic acid (EDTA) and 0.5 mM glycoletherdiamine-N,N,N'N'-tetraacetic acid (GEDTA) increased the activity in the presence of 10 mM magnesium ion. Sonication of the enzyme solution and addition of high concentration of detergent, such as Triton X-100 and Tween 20, markedly decreased the activity. Porcine liver phosphatidylcholine, phosphatidylethanolamine, and especially polyenephosphatidylcholine increased CPT activity of the arterial wall, while lysophosphatidylcholine was strongly inhibitory. The properties of arterial CPT activity under various conditions are discussed.
 
Article
The effect of CH-123 (3-carbethoxy-6-methyl-1-9-(carboxy-methyl)-1-4-oxo-6,7,8,9-tetrahydro-4H-pyrid o(1,2a)pyrimidine) was investigated on the activity of 4 lysosomal enzymes: beta-glucuronidase, beta-galactosidase, N-acetyl-beta-glucosaminidase and acid phosphatase obtained from aortic smooth muscle and liver cells of rabbits. Animals were fed on a 2% cholesterol diet for 4 weeks and used an experimental atherosclerotic group. In drug-treated groups, after 4 weeks of cholesterol feeding the diet was changed to regular food and the animals were treated daily either with 50 mg/kg CH-123 or with 250 mg/kg Clofibrate. The postnuclear supernatant of homogenates of liver and aortic cells was isolated, lysosomes were fractionated by sucrose density gradient centrifugation, and the activity of enzymes was measured. In cholesterol-fed animals the enzyme activities of aorta and liver was 3-5 times higher than in the control, i.e. in the group of rabbits fed regular food. On Clofibrate treatment the enzyme activities were 2-3 times higher, but on treatment with CH-123, they were only 1.2-1.8 times above the control. Experiments suggest that CH-123 treatment suppresses the elevated lysosomal marker enzyme activities in aortic and liver cells of atherosclerotic animals.
 
Article
The intravascular metabolism of sterol labeled [1,2,6,7-3H(N)]cholesteryl oleate and acyl labeled cholesteryl [9,10-3H]oleate and cholesteryl [1-14C]oleate was compared in the rat, an animal species without plasma cholesteryl ester transfer activity (CETA). In a first series of studies, the metabolism of sterol labeled [1,2,6,7-3H(N)]cholesteryl oleate and acyl labeled cholesteryl [1-14C]oleate was compared, and the two tracers had identical plasma clearance rates when incorporated into human low density lipoproteins (LDL). The 3H sterol labeled cholesteryl ester (CE), however, had a plasma clearance rate lower than the 14C acyl labeled CE when incorporated into rat alpha- and beta-migrating LDL and human or rat high density lipoproteins (HDL). Unesterified 3H cholesterol reappeared in the plasma whereas the 14C radioactivity in the plasma remained associated with the CE. In a second set of studies, LDL and HDL were radiolabeled with cholesteryl [9,10-3H]oleate and cholesteryl [1-14C]oleate. Large amounts of 3H radioactivity that were dialyzable and not associated with the lipoprotein CE reappeared in the plasma during the kinetic studies. The two tracers had identical plasma disappearance rates when the plasma samples were dialyzed. The results of these studies indicate that the nature of the tracer used to trace lipoprotein CE can affect the estimated kinetic parameters of plasma CE.
 
Partial nucleotide sequences of the rat VEGF gene. Putative boxes for VDRE are indicated as solid boxes and labeled Box1, Box2 and Box3. An arrow indicates the upstream ATG codon. 
Induction of VEGF promoter activity by 1,25(OH) 2 D 3 . 293 cells were transfected with a construct containing the VEGF promoter inserted upstream of the luciferase gene reporter vector pGL3-Basic. (A) Cells co-transfected with VDR expression plasmid, RXR expression plasmid or both. Cells were then incubated with 1,25(OH) 2 D 3 (V), 
Article
In previous studies we have demonstrated that the active form of vitamin D (1,25(OH)(2)D(3)) increases vascular endothelial growth factor (VEGF) expression and release in vascular smooth muscle cells (VSMC) in vitro. However, the mechanism by which 1,25(OH)(2)D(3) increases VEGF production is currently unknown. In this work, we demonstrated binding of vitamin D receptor to two response elements in the VEGF promoter. We performed promoter transactivation analysis and we observed that, in 293T cells, VEGF promoter was activated after vitamin D treatment. Using site-directed mutagenesis we have shown that both response elements are important for VEGF promoter activity. Therefore, the increase in VEGF expression and secretion induced by 1,25(OH)(2)D(3) in VSMC in vitro could be explained by direct binding of the vitamin D receptor, as a transcription factor, to VEGF promoter. These results could explain part of the beneficial effects of vitamin D treatment in renal patients by a possible VEGF-mediated improvement of the endothelial dysfunction.
 
Article
Differentiation of human promyelocytic leukemic HL-60 cells with 1,25-dihydroxyvitamin D3 (D3) results in macrophages which exhibit specific and saturable receptor-mediated processing of both native and modified low density lipoprotein (LDL). Analysis of binding kinetics demonstrated that macrophages bind LDL and acetyl-LDL with similar affinities, yet possess significantly different numbers of receptors (55 +/- 6 x 10(3) LDL receptors/cell vs 79 +/- 7 x 10(3) acetyl-LDL receptors/cell). D3-induced HL-60 macrophages challenged with LDL or acetyl-LDL exhibited suppression of HMG-CoA reductase activity as well as a significant induction in the incorporation of [14C]oleate into cholesteryl ester compared with macrophages incubated with lipoprotein depleted serum. Maximum increases in ACAT activity were obtained in macrophages incubated with 25-hydroxycholesterol plus LDL or acetyl-LDL. The increase in ACAT activity in macrophages challenged with acetyl-LDL paralleled the increase in cellular cholesterol content and the increase of oil red O lipid stainable material, imparting the macrophages with a foamy appearance. The data indicate that D3-induced HL-60 macrophages are a useful model for the study of lipoprotein--macrophage interactions as related to foam cell development and atherogenesis.
 
Article
Recent studies have demonstrated that 1,3-diacylglycerol (1,3-DAG) has several metabolic advantages over triacylglycerol (TAG) in humans and in animal models despite both oils having a similar fatty acid composition. In our current study, we have examined the effects of long-term feeding of a 1,3-DAG-rich oil on the dyslipidemia and atherosclerosis in the experimental model of the diabetic apolipoprotein E (apoE)-deficient mouse that develops accelerated atherosclerosis. Diets containing 1,3-DAG-rich oil or TAG oil were administered to control non-diabetic apoE-dificient and diabetic apoE-deficient mice for 20 weeks. In diabetic apoE-deficient mice, 1,3-DAG reduced the extent of atherosclerotic lesions in the aortic arch and thoracic aorta by 37 and 44%, respectively, compared to TAG. Further, in diabetic apoE-deficient mice, plasma total cholesterol and triglyceride levels were significantly lower in the 1,3-DAG-fed group than in the TAG-fed group. This occurred partially through an apparent reduction in the size of triglyceride-rich lipoproteins but not apparently by reducing the number of lipoprotein particles. By contrast the control non-diabetic apoE-deficient mice showed no differential responses to the type of oil at least over 20 weeks. We have demonstrated that dietary 1,3-DAG-rich oil reduced atherosclerosis in diabetic apoE-deficient mice, and was associated with reduction in plasma cholesterol especially within larger triglyceride-rich lipoproteins.
 
Article
Women under hormone replacement therapy carry an increased risk of venous thromboembolism (VTE), mostly during the first year. Despite great efforts devoted to hormone therapy research, VTE remains a major drawback of estrogenic therapy, and the search for new compounds continues. We have synthesized and evaluated prolame, an aminoestrogen with anticoagulant properties. The aim of our work was to elucidate the anticoagulant mechanism of prolame. We studied the effects of prolame on nitric oxide (NO) synthesis in cultured endothelial cells and platelets using flow cytometry, on NO metabolites using a modified Griess method, on NO formation in vivo using electron paramagnetic resonance spectroscopy, on participation of nuclear estrogen receptors using flow cytometry, and on endothelial NO synthase (eNOS) mRNA expression using RT-PCR. We also studied the impact of prolame-treated endothelial cells (EC) on ADP-induced platelet aggregation, as well as the ability to prevent occlusive thrombi in an in vivo mice thrombosis model. (a) Prolame induces NO production in ECs, platelets, and in a mouse model in vivo. (b) The NO-elevating effect of prolame can only be partially attributed to the nuclear estrogen receptors (ERs) since endothelial nitric oxide synthase (e-NOS) is slightly induced (37%) in ECs treated with prolame. (c) Platelets become 60% less responsive to aggregation induced by 10muM ADP when in suspension with prolame-treated ECs. (d) Prolame reduces the formation of thrombi in an in vivo thrombosis model. Prolame could be a preferred alternative to other estrogens because of its reduced thromboembolic risk.
 
Article
Serum 1,5-anhydro-D-glucitol (1,5-AG) is well-known to be a useful clinical marker of both short-term glycemic status and postprandial hyperglycemia. In addition, previous epidemiological studies have shown that an increased postload glucose level in an oral glucose tolerance test is a risk factor for cardiovascular diseases (CVD). However, no previous prospective study has reported the association between serum 1,5-AG levels and the risk of CVD. In this study, we examined whether serum 1,5-AG levels can predict the incidence of first-ever CVD. Our study was a population-based cohort study in an urban area of Japan. Study subjects comprised 2095 initially healthy Japanese (991 men and 1104 women, mean age: 58.5 years) with no history of coronary heart disease (CHD) or stroke. They were followed up for an average of 11.1 years, and 147 CVD events (64 CHD and 83 strokes) were observed. The adjusted hazard ratios (HRs) of all CVD in men increased linearly (p=0.004). The HR in the category with serum 1,5-AG levels of 14.0 μg/mL or less was 2.22 (95% confidence interval; 1.24-3.98) compared to the reference category (24.5 μg/mL or greater). Similar results were also shown with a sensitivity analysis in non-diabetic men. Conversely, no significant relationship between serum 1,5-AG levels and CVD risks was observed in women. Our results suggest that measurement of serum 1,5-AG levels is useful to detect individuals, especially men, at higher risk for CVD, regardless of the presence or absence of diabetes.
 
Article
An analysis of blood groups and cholesterol has been made as part of a longterm prospective investigation of ischemic heart disease among 10,000 Israeli males, aged 40 and over. The total mean serum cholesterol level for men of blood group A1 was found to be significantly higher than the mean for the combined ABO groups, although this did not hold for every individual country of origin. Among the Rh, MN, Kell, Duffy, and Kidd blood groups, Kell was the only one which showed a significant association with mean serum cholesterol. Men with both A1 and Kell+ exhibited a higher mean cholesterol level than either group separately. The question is raised whether the significant statistical associations in this study population have biological implications.
 
Article
Abdominal obesity is associated with coronary heart disease (CHD) but its relationship to peripheral vascular disease (PVD) is under-researched. This study is to evaluate the association of PVD with two measures of abdominal obesity, waist-to-thigh ratio (WTR) and waist circumference (WC). The study population consisted of 5057 adults aged 40 years or older who participated in NHANES 1999-2002. PVD was defined as an ankle-brachial index (ABI) <0.9. WTR and WC were divided into gender-specific quartiles. Of the 5057 participants, 367 (7.3%) were identified as having PVD. The cross-sectional associations were evaluated using multiple logistic regressions separately by gender. After adjusting for demographic variables and traditional risk factors of PVD, WTR was strongly associated with PVD in men. The odds ratios (95% CI) across the WTR quartiles were 4.21 (1.86,9.51), 4.44 (2.17,9.09) and 4.68 (2.13,10.32) compared to the 1st quartile (P for trend=0.018). Among women, strong linear trend was found indicating significant association between WTR and PVD (P for trend=0.007). While WC was not associated with PVD among men, among women, those in the 4th quartile of WC had an increased odds of PVD compared to the 1st quartile (odds ratio: 2.94, 95% CI: 1.01-8.88). WTR is associated with PVD in men and women. In contrast, WC is associated with PVD in women, but not in men. Well-controlled prospective studies are needed to assess these two measures of body fat distribution and its association with the development of PVD in men and women.
 
Article
The hypocholesterolemic and anti-atherogenic properties of sulfamic acid ((2,4,6-tris (1-methylethyl) phenyl) acetyl) 2,6-bis(1-methylethyl) phenyl ester, the ACAT inhibitor, CI-1011, was tested in 120 male F1B hamsters fed a hypercholesterolemic chow-based diet containing 10%, coconut oil and 0.05% cholesterol plus: (i) no drug treatment (HCD); (ii) 3 mg/kg per day (HCD+3): (iii)10 mg/kg per day (HCD+10); (iv) 30 mg/kg per day (HCD+30) of CI-1011; or (v) 500 mg/kg per day of cholestyramine (CSTY). Plasma samples were collected at 8 and 10 weeks for measurement of total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG). For the progression studies, animals were euthanized after 10 weeks for aortic fatty streak area and hepatic cholesterol analysis. For the regression study, a cohort of the HCD was treated with 30 mg/kg per day of CI-1011 (regression) for an additional 8 weeks. The HCD+3, HCD+10, HCD+30 and CSTY lowered plasma TC (25, 32, 34 and 32%, respectively), VLDL-C (62, 74, 71 and 75%, respectively), LDL-C (25, 38, 47 and 46%, respectively) and TG (48, 47, 42 and 45%, respectively). All treatments resulted in a significant lowering of aortic fatty streak area (68, 86, 93 and 94%, respectively) and reduction in hepatic cholesteryl esters (57, 65, 67 and 70%, respectively). Regression of aortic fatty streak area was 90% after 8 weeks of HCD+30 treatment. Also during the regression phase, plasma TC, LDL-C and TG were lowered 23, 33 and 47%, respectively, as well as, hepatic cholesteryl esters (76%). Significant correlations between plasma LDL-C concentration and aortic fatty streak area (r=0.62, P < 0.004) in the HCD+10 group, suggest that CI-1101 altered aortic lipid infiltration primarily by its effect on plasma lipids. However the 30 mg/kg per day dose of CI-1011 which additionally reduced aortic fatty streak area by 51% relative to the 10 mg/kg per day dose was only associated with a 14% further decrease in plasma LDL-C. Finally the 10-fold regression of aortic fatty streak area was associated with only a 35% reduction in plasma LDL-C. These exceptions to the lipid-lesion relationship raise the possibility of additional effects of CI-1011, which may occur independent of or in concert with lipoprotein cholesterol lowering. It is concluded that in hypercholesterolemic hamsters, CI-1011 is approximately 50 times more potent than cholestyramine in cholesterol-lowering, reduction and regression of aortic fatty streak area.
 
Article
Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors have been shown to reduce atherosclerotic lesions in animals; however, the mechanism(s) for this effect remains unclear. Therefore, we used cultured primary human monocyte-derived macrophages (HMMs) to examine the effect of the ACAT inhibitor, avasimibe (CI-1011), during foam cell formation and during cholesterol efflux from established foam cells. To examine the effect of CI-1011 on foam cell development, HMMs were incubated with aggregated acetylated LDL (ag-acLDL)+/-CI-1011 for 48 h. Total cholesterol (TC) was 29% lower in HMMs incubated with ag-acLDL and CI-1011 compared with ag-acLDL (P<0.05). To determine if TC reduction was due to reduced ag-acLDL uptake by CI-1011, 125I-acLDL binding at 4 degrees C for 4 h to HMMs preincubated with acLDL or ag-acLDL, CI-1011, acLDL+CI-1011, or ag-acLDL+CI-1011 for 48 h was measured. Specific binding was 40% lower in cells preincubated with acLDL+CI-1011, 52% lower in cells preincubated with ag-acLDL+CI-1011 and 49% lower in cells preincubated with CI-1011 compared with cells preincubated with acLDL (P<0.0003). Because CI-1011 appeared to directly affect acLDL binding, 125I-acLDL (3-80 microg protein/ml) binding was done in HMMs preincubated with CI-1011 (0-10 microg/ml) for 48 h. The calculated B(max) decreased in HMMs exposed to increasing concentrations of CI-1011, suggesting that CI-1011 altered scavenger receptor function and/or number. To examine the effects of CI-1011 on cholesterol efflux from established foam cells, we first examined whether CI-1011 was cytotoxic. HMMs were preincubated with ag-acLDL for 24 h, and then radiolabeled with [14C]adenine for 2 h (time zero). The radiolabeled cells were exposed to control RPMI medium or the same medium+HDL, CI-1011, or HDL+CI-1011 for 24 h. The release of [14C]adenine into the medium was not significantly different between cells exposed to RPMI, HDL, CI-1011, or HDL+CI-1011, suggesting that CI-1011 was not cytotoxic. Foam cells exposed to RPMI and CI-1011 (1-10 microg/ml) for 48 h showed time dependent reduction in cellular TC mass, with a corresponding increase in radiolabeled unesterified cholesterol into the medium. We then asked whether CI-1011 enhanced apoE mediated cholesterol efflux. Although cellular apoE increased between 2- and 7-fold in foam cells compared to control macrophages, apoE secreted into the medium was not significantly different between cells exposed to RPMI or CI-1011. Thus, CI-1011 exerted anti-atherogenic effects by reducing TC accumulation, inhibiting acLDL binding, and by limiting lipid storage in HMMs.
 
Article
Lipoprotein(a) (Lp(a)), which is generated through the covalent association of apolipoprotein(a) (apo(a)) and apo B-100-LDL, is an independent risk factor for several vascular diseases. Therefore, there is interest in developing therapies for lowering Lp(a). This investigation was carried out to determine the effect of CI-1011, a potent lipid regulator in rodents, on Lp(a) and other lipid parameters in cynomolgus monkeys (Macaca fascicularis). Nine healthy male monkeys on a normal chow diet were orally treated with CI-1011 at 30 mg/kg per day for 3 weeks. Lp(a) and total cholesterol levels were significantly decreased after 1 week and maximally reduced to 68 and 73% of control levels, respectively, after 3 treatment weeks. The decreases in total cholesterol were mainly due to changes in low density lipoprotein (LDL). The LDL:HDL ratio decreased by 30%. Triglycerides were unaffected by treatment. Lp(a) and total cholesterol levels returned to pretreatment values after stopping treatment suggesting a direct effect of the compound on their inhibition. Further studies demonstrated that CI-1011 was effective at a low dose of 3 mg/kg per day after 1 week of administration. CI-1011 also decreased apo B-100 to 80% of control levels, but this change was not sufficient to account for the Lp(a) lowering. There was also no correlation between the changes in Lp(a) and apo B-100 levels. Treatment of cynomolgus monkey primary hepatocyte cultures with CI-1011 resulted in a dose-dependent inhibition of Lp(a) levels suggesting a direct hepatic effect of the compound. Western blot analysis of the samples showed that changes in Lp(a) were associated mainly with decreased apo(a) (47%), but not apo B-100 (17%). These results demonstrate that CI-1011 effectively decreases Lp(a) levels both in vivo and in vitro.
 
Article
A C1019T polymorphism in the human connexin37 (hCx37) gene has been associated with cardiovascular risk, but it remains debatable whether the 1019C or the 1019T allele carries this risk. Here, we investigated whether these allelic variants are differentially predictive of increased risk for coronary artery disease (CAD) and myocardial infarction (MI). A total of 781 Swiss participants, including 597 patients diagnosed with CAD, 50% who reported previous MI, and 184 control subjects were genotyped. Patients in the +CAD group had a higher frequency of the Cx37-1019C allele (70.3% versus 65.0%, p=0.004). Multivariate analysis showed that the hCx37-C1019T polymorphism is an independent predictor of CAD (odds ratio=2.13, confidence interval=1.31-3.46 and p<0.01). Moreover, this polymorphism is not associated with any of the other characteristics examined, including gender, age, body-mass-index, diabetes, total/HDL/LDL-cholesterol, triglycerides, apoA-I, apoB, hypertension and cigarette smoking. In comparison with the -CAD group, we observed an increase of the Cx37-1019C allele in the +MI +CAD subgroup (71.2% versus 65.0%, p=0.002) but not in the -MI +CAD subgroup. Allelic frequency comparisons of these three subgroups predicted that this polymorphism is also an independent risk factor for MI. In conclusion, our results reveal the importance of screening the Cx37-1019C allele for both CAD and MI risk assessments.
 
Article
Elevated urinary albumin excretion (UAE) is associated with the risk of cardiovascular disease (CVD) and all-cause mortality. We tested the hypothesis that elevated UAE improves cardiovascular risk stratification in an elderly cohort aged 68-102 years. We evaluated UAE in 3112 participants of the Cardiovascular Health Study who attended the 1996-1997 examination and had median follow up of 5.4 years. Elevated UAE was defined as urinary albumin to creatinine ratio > or =30 microg/mg. Microalbuminuria and macroalbuminuria were defined as urinary albumin to creatinine ratio 30-300 microg/mg and >300 microg/mg, respectively. Outcomes included CVD (myocardial infarction, stroke, cardiovascular death) and all-cause mortality. Cox proportional hazards models were used to assess the risk of outcomes associated with elevated UAE. The prevalence of elevated UAE was 14.3%, 17.1% and 26.9% in those aged 68-74, 75-84 and 85-102 years, respectively. CVD incidence and all-cause mortality were doubled (7.2% and 8.1% per year) in those with microalbuminuria and tripled (11.1% and 12.3% per year) in those with macroalbuminuria compared to those with normal UAE (3.3% and 3.8% per year). The increased CVD and mortality risks were observed in all age groups after adjustment for conventional risk factors. The adjusted population attributable risk percent of CVD and all-cause mortality for elevated UAE was 11% and 12%, respectively. When participants were cross-classified by UAE and Framingham Risk Score categories, the 5-year cumulative incidence of coronary heart disease among participants with elevated UAE and a 5-year predicted risk of 5-10% was 20%, substantially higher than 6.3% in those with UAE <30m microg/mg. Elevated UAE was associated with an increased risk of CVD and all-cause mortality in all age groups from 68 to 102 years. Combining elevated UAE with the Framingham risk scores may improve risk stratification for CVD in the elderly.
 
Article
A biochemical, histologic and morphometric evaluation of spontaneous, diet-induced (thoracic aorta) and injury-induced (iliac-femoral) atherosclerotic lesions was performed in rabbits maintained on varying levels of dietary cholesterol. Rabbits were meal-fed a 3% peanut oil, 3% coconut oil diet containing 0%, 0.1%, 0.25%, 0.5%, 1.0%, 1.5% or 2.0% cholesterol for 9 weeks. Plasma total cholesterol exposure (area under cholesterol-time curve (TC-AUC)) increased diet-dependently over the course of the study. VLDL and LDL cholesterol (VLDL-C, LDL-C) comprised 41% and 55%, respectively, of the plasma total cholesterol at cholesterol levels > 700 mg/dl (TC-AUC > 31,868 mg day/dl) and both VLDL-C and LDL-C were linearly related to TC-AUC (r = 0.98). Plasma TC-AUC was linearly related to thoracic aortic cholesteryl ester (CE) content (r = 0.74) and thoracic aortic lesion coverage (r = 0.66). In the injury-induced iliac-femoral lesion, plasma TC-AUC was linearly related to both iliac-femoral CE content (r = 0.80) and macrophage/lesion ratio (r = 0.64). At plasma cholesterol levels greater than 700 mg/dl, CE content of the iliac-femoral lesion ranged from 35 to 69 micrograms/mg dry defatted tissue, > 75% of the lesions were fibrofoamy in nature and macrophage/lesion area ratio was 0.46 to 0.55 while lesion area remained constant. VLDL-C and LDL-C were highly correlated with the CE content of both thoracic and iliac-femoral lesions, thoracic aortic lesion coverage and macrophage/lesion area ratio (r = 0.86-0.99). We conclude that the composition, extent and type of atherosclerotic lesion induced in rabbits is dependent upon the overall plasma cholesterol exposure, VLDL and LDL cholesterol content and whether lesions are induced by diet alone or both diet and chronic endothelial injury. In addition, various stages of atherosclerotic lesion formation can be replicated in the rabbit by titrating the animal's overall plasma cholesterol exposure.
 
Article
The hypolipidemic effect of NK-104 and its mechanisms of action (effects on hepatic sterol synthesis, low density lipoprotein (LDL)-receptor expression and very low density lipoprotein (VLDL) secretion) were studied in guinea pigs using simvastatin as a reference substance. There was a dose-dependent and significant reduction of both plasma total cholesterol (17.4, 24.5 and 45.3% at 0.3, 1 and 3 mg/kg, respectively) and triglycerides (21.1 and 32.2% at 1 and 3 mg/kg, respectively) after 14-day administration of NK-104. Simvastatin at 30 mg/kg lowered plasma total cholesterol (25.0%) but not triglyceride levels. NK-104 (3 mg/kg) and simvastatin (30 mg/kg) inhibited hepatic sterol synthesis by approximately 80%, 3 h after dosing, and enhanced LDL receptor binding-capacity of liver membranes 1.5-fold after 14-day dosing. The former group accelerated LDL clearance somewhat more markedly than the latter, and increased fractional catabolic rate 1.8-fold (vs. 1.4-fold). Furthermore, only the NK-104 (3 mg/kg) suppressed VLDL secretion into the liver perfusate (triglyceride. 19.9%; apoB, 24.2%) with extensive reduction of hepatic sterol synthesis caused by prolonged action. These results indicate that NK-104 and simvastatin at 10 times the dosage of the former, similarly enhances hepatic LDL receptor; however, only NK-104 with prolonged action suppresses VLDL secretion to show higher cholesterol-lowering potency and triglyceride-reducing effect.
 
Article
Intracellular cholesterol biosynthesis may play a key role in supplying cholesterol (as cholesteryl ester) for the neutral core of very low density lipoprotein (VLDL), thus modulating the secretion of apolipoprotein B-100 (apo B-100) from hepatocytes. The effect of compound NK-104 was studied, a new competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA-reductase), on apo B-100 synthesis and secretion from the human hepatoma cell line Hep G2. Cells were preincubated with NK-104 (0.01-5 microM) in the presence or absence of oleate (0.8 mM). Apo B-100 in the medium was determined by an enzyme-linked immunosorbent assay (ELISA). Incubation of Hep G2 with NK-104 resulted in a marked inhibition of cholesterogenesis (up to 95%), determined as incorporation of [14C]acetate into sterols, and decreased in a dose-dependent manner apo B-100 secretion, both in basal conditions (from 110 to 82 ng/mg cell protein, P < 0.01) and after incubation with oleate (from 227 to 165 ng/mg cell protein, P < 0.01). Density gradient for distribution of apo B-100 secreted, showed that this decrease was essentially due to a reduction of apo B-100 associated with lipoproteins in the density range of low density lipoproteins (LDL). Pulse chase experiment demonstrated that NK-104 did not affect the synthetic rate of apo B-100 but increased intracellular degradation of newly synthesized protein. The compound had only marginal effect on the mass of intracellular triglyceride but significantly decreased intracellular mass of free cholesterol and cholesteryl ester (P < 0.01). It is speculated that the ability of compound NK-104 to decrease apo B-100 secretion from Hep G2 cells is due to a decreased intracellular cholesterol availability.
 
Article
The clinical efficacy and safety of pitavastatin (NK-104), a novel HMG-CoA reductase inhibitor, during long-term treatment, were examined in 25 patients (male/female=11/14, mean age=53+/-13 (mean+/-SD) years) with heterozygous familial hypercholesterolemia (FH). After a period on placebo of >4 weeks, 2 mg/day of pitavastatin was administered for 8 weeks, and the dose was increased to 4 mg/day for up to 104 weeks. Total cholesterol (TC) decreased by 31% from the initial value of 340+/-57 to 237+/-40 mg/dl (P<0.0001) at week 8. During treatment with the higher dose, TC decreased even further to 212+/-35 mg/dl at week 12; it decreased by 37% from the initial value (P<0.0001). Similarly, the baseline low-density lipoprotein (LDL)-cholesterol (LDL-C) decreased by 41% at week 8, and by 49% at week 12, from 267+/-61 mg/dl at baseline. These findings indicate a dose-dependent effect of the drug on TC and LDL-C concentrations. To examine whether the levels of circulating matrix metalloproteinases (MMPs) and their endogenous inhibitors (tissue inhibitors of metalloproteinases: TIMPs) are altered during lipid-lowering therapy, we also measured their plasma levels. The mean levels of MMP-2 and -3 were significantly increased. No significant alteration was found in MMP-9, TIMP-1 and -2 levels. As for the safety of pitavastatin, adverse reactions were observed in one case (4%) of subjective and objective symptoms. The effects of pitavastatin on TC and LDL-C were stable during long treatment of patients with heterozygous FH.
 
Article
Previous studies from this laboratory have shown that degradation of heparan sulphate proteoglycan by both living macrophages and macrophage lysosomal heparanase induces phenotypic change of vascular smooth muscle cells (SMC) from a high volume fraction of myofilaments (V(v)myo) to a low V(v)myo [Campbell et al. Exp Cell Res 1992; 200: 156-167]. The aim of this study was to determine whether matrix metalloproteinase (MMP) activity is also involved in the induction of SMC phenotypic change by macrophages. A specific inhibitor of MMPs (BB94) was able to block macrophage-induced SMC phenotypic change and subsequent DNA synthesis in freshly dispersed SMC seeded in primary culture at confluent density. The inhibitor did not block these SMC changes when SMC were seeded at low density without macrophages nor did it block heparanase activity directly. We also determined whether heparanase and MMP activities are upregulated together in vivo. Artery homogenates were analysed in a heparanase enzyme assay and for MMPs using zymograms. Increased heparanase activity was observed 3-14 days following balloon catheter injury of rabbit carotid arteries, and returned to control levels 6 weeks after injury. Active MMP2 was induced with heparanase after injury. MMP9 induction was also apparent 6 h after injury. Immunohistology on sections of these arteries showed the presence of MMPI1, 2, 3 and 9 with these MMPs being strongly induced in the intima 7 days after balloon catheter injury. Both heparanase and MMP activities were also present in human end-stage complex lesions from coronary arteries, carotid endarterectomies and abdominal aortic aneurysms. Because MMPs and heparanase are expressed at the same time, it is possible that MMPs facilitate heparanase activity in promotion of phenotypic modulation of SMC in vivo during neointimal thickening following injury and in atherosclerotic lesions.
 
Article
The antioxidant AGI-1067 was shown to reduce experimental atherosclerosis. The present study originally intended to study restenosis as a primary endpoint but was subsequently modified to primarily investigate the effects of AGI-1067 on coronary atherosclerosis. This placebo-controlled randomized trial assessed the effects of AGI-1067 280 mg qd started before percutaneous coronary intervention (PCI) and administered for 12 months after PCI on atherosclerosis progression as assessed by coronary intravascular ultrasound (IVUS). Among patients with IVUS examinations considered technically adequate both at baseline and follow-up upon central laboratory assessments (n=232), plaque volume was not significantly modified with placebo (least squares mean change: -0.4mm(3), P=0.85 versus baseline), but was significantly reduced by -4.0mm(3) at end of treatment in the AGI-1067 group (P=0.001 versus baseline, P=0.12 versus placebo). LDL-cholesterol varied by -9% and +4% in the placebo and AGI-1067 groups, respectively (P<0.05 between groups), and HDL-cholesterol was reduced by 1% with placebo and 14% with AGI-1067 (P<0.05 between groups). Plasma myeloperoxidase was reduced by 6% with AGI-1067 (P<0.05) but hs-CRP was not significantly different between groups. Atherosclerosis regression (-4.0mm(3)) was observed in patients treated with AGI-1067, although this was not significantly different from placebo. The anti-inflammatory effect of AGI-1067 is supported by reduced levels of myeloperoxidase.
 
Article
Apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) represent 80 90% of the protein content of high density lipoproteins (HDL). Previously we have identified a Finnish family with an apo A-I variant (Lys107-->0) associated with reduced plasma HDL cholesterol level and decreased lipoprotein (Lp)(AI w AII) concentration compared to unaffected family members. To determine the in vivo metabolism of apo A-I and apo A-II in the carriers of apo A-I (Lys107-->0) variant we radioiodinated normal apo A-I with 125I and apo A-II with 131I and compared the kinetic data of two heterozygous apo A-I(Lysl07-->0) patients (HDL cholesterol leves 0.31 and 0.69 mmol/l) to that of eight normolipidemic, healthy control subjects. Plasma radioactivity curves of 125I-labelled normal apo A-I of the patients demonstrated accelerated clearance of apo A-I compared to control subjects. In the two patients the fractional catabolic rates (FCR) of apo A-I were 0.347/day and 0.213/day, respectively, while the mean FCR of apo A-I of the control subjects was 0.151 +/- 0.041/day. Similarly, the plasma decay curves of the 131I-labelled apo A-II showed more rapid clearance of apo A-II in the two patients than in control subjects. The FCR of apo A-II in the two patients were 0.470/day and 0.234/day, while the mean FCR of apo A-II in control subjects was 0.154 +/- 0.029/day. The calculated production rates of apo A-I were similar in patients and in control subjects, and the production rates of apo A-II were significantly higher in patients than in control subjects. Our results show that the Lp(AI w AII) deficiency in patients with the apo A-I(Lys107-->0) is associated with increased fractional catabolic rates of normal apo A-I and apo A-II, while the production rates of these apolipoproteins are normal (apo A-I) or slightly increased (apo A-II).
 
Article
Epidemiological surveys show decrease or reversal of male predominance in cardiovascular mortality in the very old, but the actual condition of atherosclerosis in the very old is largely unknown. The objective of this paper is to reveal whether the atherosclerosis continues to progress, or the gender-related difference exists in the very old. The subjects were 1074 consecutive autopsy cases of in-hospital death. The male:female ratio was 1.1:1 and the average age was 80 years. Macroscopic evaluation was performed on the degree of atherosclerosis in 10 arteries including the intracranial arteries, carotid artery, aorta, coronary artery, and femoral artery. The severity of atherosclerosis differed greatly among arteries. The age-related increase of the atherosclerotic degree was evident, even after 80 years of age. The atherosclerosis was more severe in males than in females in their 60s, but this male predominance decreased with ageing and finally disappeared in their 90s. The sustained progression of atherosclerosis and loss of the gender-related difference probably account for the increase of cardiovascular mortality in very old females. They also suggest that the prevention of the atherosclerotic progression is still important in the seventh and eighth decade of life.
 
Article
Eight hundred and twenty-three Chinese Han adults aged over 40, including 466 men and 357 women were enrolled in the study to examine the association of +1075C and -480T polymorphisms in hepatic lipase gene with plasma lipoprotein and apolipoprotein levels. In this population the allele frequencies for HL +1075C and minus -480T were 0.053 and 0.362, respectively and the prevalence of HL +1075C/C and -480T/T were 0.006 and 0.132, respectively. Overall, the normal HDL-C (> or = 35 mg/dl) subjects had a higher carrier frequency of HL +1075C than the low HDL-C (<35 mg/dl) subjects (0.108 vs 0.029, P=0.039). However, when tested separately, the carrier frequency of HL +1075C was not significantly different between normal and low HDL-C females (0.101 vs 0.083, P=0.843). In males, the normal HDL-C subjects had a higher carrier frequency of HL +1075C than the low HDL-C subjects (0.113 vs 0.018, P=0.026). No significant difference of frequencies of HL -480T genotypes -480T/T,-480C/T and -480C/C was found between normal and low HDL-C subjects. Among plasma TG, TC, HDL-C, apo AI, apo AII, apo B100, apo CII, Apo CIII and apoE, only HDL-C and apo AI were significantly different among the three genotypes +1075A/A,+1075A/C and +1075C/C in men (P=0.029 and 0.032). No association was found in women. Male subjects with CC had a higher HDL-C than those with AC (P=0.020) and AA (P=0.013), AC higher than AA (P=0.017). Male subjects with CC had a higher apo AI than AC (P=0.013) and AA (P=0.019), AC higher than AA (P=0.021). Although not so significant (P=0.053) as HDL-C and apo AI, male subjects with CC had a higher apo AII than those with AC and AA. No significant difference of lipoprotein and apolipoprotein traits was found among the three -480T genotypes -480C/C,-480C/T and -480T/T, in the sample overall and in men and women separately. These results indicate that HL +1075C, not -480T polymorphism is associated with plasma high density lipoprotein cholesterol and apolipoprotein AI in men in this Chinese population.
 
Article
A correlation between atherogenesis and lipoprotein oxidation was first suggested by experiments showing increased uptake by macrophages of oxidized LDL and oxidized LDL injury to cultured cells. Recent data which demonstrate the existence of oxidized lipoproteins in vivo, combined with studies showing a 'protective' effect of antioxidants against atherosclerosis progression, have greatly increased the interest in theories posing that lipoprotein oxidation is causally related to arterial disease. The fact that dozens of new compounds are produced upon the oxidation of low density lipoprotein has led, perhaps not surprisingly, to numerous discoveries in vitro of altered cell function induced by exposure of cells to oxidized LDL that are distinct from those resulting from exposure to native LDL. This brief overview will describe selected altered cell functions of oxidized lipoproteins and how they may impact on atherosclerosis.
 
Article
The risk of cardiovascular disease (CVD) in SLE patients is very high. It is therefore surprising that IL-10 has been discussed both as pathogenic in SLE and as an atheroprotective cytokine. In contrast, TNF is believed to be atherogenic and we recently reported that raised activity in the TNF-system is implicated in SLE-related CVD. Twenty-six (aged 52 +/- 8 years) female patients with SLE and a history of CVD (myocardial infarction, angina, stroke or claudication) were compared with 26 age-matched SLE patients without CVD (SLE controls) or 26 age-matched population controls. The -1087IL-10 gene polymorphism was determined by PCR with restriction endonuclease mapping. Serum IL-10 and TNF-levels were determined by ELISA. The A allele frequency of -1087IL-10 gene in SLE/CVD was higher than in SLE controls (0.62 versus 0.42, p < 0.05). Ten (38%) of 26 SLE/CVD exhibited IL-10 AA genotype compared with five (19%) of 26 SLE controls. Serum IL-10 and TNF-levels were raised in SLE/CVD compared with SLE controls or population controls (p < 0.001). Furthermore, in SLE/CVD, a significantly reduced IL-10:TNF ratio was observed in patients with IL-10 AA genotype compared with AG or GG genotype (0.56 versus 0.77 versus 1.24, p < 0.05). In SLE controls and population controls, individuals with IL-10 GG genotype tended to have higher IL-10:TNF ratio. In conclusion, the A-1087IL-10 allele which has been reported to cause a lower capacity for IL-10 production could contribute to CVD in SLE. Furthermore, the IL-10 AA genotype is associated with reduced ratio of atheroprotective to atherogenic cytokines in SLE patients with CVD.
 
Article
Hydroxy-methyl-glutaryl co-enzyme A (HMG-CoA) reductase inhibitors or statins are well tolerated by most patients, but can produce a variety of skeletal muscle problems including myalgia, creatine kinase (CK) elevations and clinically important rhabdomyolysis. We have previously demonstrated that the CK response to downhill walking is greater in statin compared to placebo treated subjects. This study examined the CK response to downhill walking in subjects treated with low and high dose of atorvastatin. 79 subjects with LDL cholesterol>100mg/dL were randomly assigned to atorvastatin 10mg (N=42) or 80 mg (N=37) for 5 weeks. Subjects performed a downhill walking exercise during the fifth week of treatment. Leg muscle soreness, plasma CK and CK-MB levels were measured daily for 4 days following the exercise. CK, CK-MB and muscle soreness increased above pre-exercise levels in all subjects after the exercise. There were no differences in the CK, CK-MB or soreness response between the high and low dose treatment groups at any time point. The downhill walking model of muscle injury does not distinguish between high and low dose atorvastatin therapy either because this test is insensitive to differences among statin doses or because there is no difference in muscle injury between these two drug doses with this statin. Clinicians should be aware, however, that exercise can increase CK levels with even low dose statin therapy.
 
Article
Reduction in atherosclerosis has been reported in experimental animals fed mixtures of conjugated linoleic acid (CLA). In this study, the major naturally occurring CLA isomer (cis-9,trans-11) was tested in an atherosclerosis-prone mouse model. In a model of insulin deficient apoE deficient mice, 16 animals were fed for 20 weeks with supplemental CLA (09.%, w/w) and compared with a similar number of mice of this phenotype. A control comparison was made of metabolic changes in non-diabetic apoE deficient mice that develop little atherosclerosis over 20 weeks. At 20 weeks, plasma lipids were measured and aortic atherosclerosis quantified by Sudan staining in the arch, thoracic and abdominal segments. The diabetic apoE deficient mice developed marked dyslipidemia, primarily as cholesterol-enriched chylomicron and VLDL-sized lipoproteins and atherosclerosis in the aortic arch. However, there were no significant differences between CLA fed and non-CLA fed mice in either phenotype in plasma cholesterol concentration (in diabetic: 29.4+/-7.7 and 29.5+/-5.9 mmol/L, respectively) or in the area of aortic arch atherosclerosis (in diabetic: 24.8+/-10.3 and 27.6+/-7.7%, respectively). However, among diabetic mice the triglyceride concentration in triglyceride-rich lipoproteins was significantly lower in those fed CLA (for plasma 2.2+/-0.8 to 1.1+/-0.3 mmol/L; P<0.001), a significant difference that was seen also in the non-diabetic mice in which HDL cholesterol increased significantly with CLA (0.35+/-0.12-0.56+/-0.15 mmol/L). In this atherosclerosis-prone model, the diabetic apoE deficient mouse, supplemental 0.9% CLA (cis-9,trans-11) failed to reduce the severity of aortic atherosclerosis, although plasma triglyceride concentration was substantially lowered and HDL cholesterol raised.
 
Article
Human vascular smooth muscle cells (VSMC) are a component of blood vessels, and secrete a variety of cytokines in atherosclerotic loci. Interleukin-11 (IL-11), a member of IL-6-like cytokines, is reported to be involved in inflammation and tissue remodeling, both of which are observed in atherosclerosis. However, no information is available as to the production of IL-11 by VSMC. Therefore, the expression of IL-11 in VSMC is investigated. The amounts of IL-11 protein and mRNA were determined by enzyme-linked immunosorbent assay (ELISA) and Northern blot analysis, respectively. The expression of IL-11 in VSMC was also immunohistochemically determined. IL-1 alpha, transforming growth factor-beta (TGF beta) and, to a lesser extent, tumor necrosis factor-alpha (TNF alpha) stimulated the IL-11 production by VSMC, and the stimulatory effects of IL-1 alpha and TGF beta on IL-11 production were dose-dependent. IL-1 alpha and TNF alpha synergistically augmented TGF beta-stimulated IL-11 production by VSMC. Immunohistochemical staining also revealed the expression of IL-11 protein in VSMC. Furthermore, IL-1 alpha, TGF beta, and TNF alpha induced IL-11 gene expression in VSMC. Because IL-6-like cytokines are reported to be cytoprotective, monokine-stimulated IL-11 may have a potent protective role in atherosclerotic lesions.
 
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There is limited knowledge on drinking patterns and the progression of atherosclerosis. Previous studies have shown conflicting results between alcohol consumption and atherosclerotic progression. We investigated the association between the pattern of binge drinking and the 11-year progression of carotid atherosclerosis in a population-based sample of middle-aged men. This study is a part of the FinDrink Study, based on the Kuopio Ischemic Heart Disease Risk Factor Study. We investigated the effect of drinking patterns on the changes in maximum and mean intima-media thickness, including changes in maximum plaque height in 751 participants as measured by carotid ultrasound. The increased atherosclerosis progression was statistically significant among those men with binge drinking of >or=6 drinks per session (22.4% of total number of participants) using different covariates in different models according to the mean increase in maximum intima-media thickness (Model 1, p=0.008; Model 2, p=0.031, Model 3, p=0.037) and the mean increase in maximum plaque height (Model 1, p=0.002; Model 2, p=0.012, Model 3, p=0.017). Our study shows that binge drinking was associated with an increased atherosclerosis progression during an 11-year follow-up in middle-aged men, independent of the total alcohol consumption.
 
Article
Leukocyte telomere length shortening has recently been associated with type 2 diabetes mellitus (T2D). Whether this observation was modulated by genetic variation within the telomere-pathway genes remains elusive. To date, no prospective epidemiological data on the relationship of telomere-pathway gene variation with T2D are available. The association between 150 tagging-SNPs (tSNPs) of 11 telomere-pathway genes (TERC, UCP1, TERT, POT1, TNKS, TERF1, TNKS2, TEP1, ACD, TERF2 and TERF2IP) and incident T2D was investigated in 22,715 Caucasian female participants of the prospective Women's Genome Health Study. All were free of known cardiovascular disease, cancer and diabetes at baseline. During a 13-year follow-up period, 1445 participants developed an incident T2D. Multivariable Cox regression analysis was performed to investigate the relationship between genotypes and T2D risk assuming an additive genetic model. Haplotype block analysis was also performed. A total of eleven tSNPs within TERF1, TNKS, TEP1, ACD, and TERF2 were associated with T2D risk (all p-uncorrected <0.050). Further investigation using the haplotype-block analysis again revealed an association of several prespecified haplotypes of TERF1, and TEP1 with T2D risk (all p-uncorrected <0.040). If corroborated in other prospective studies, the present findings suggest that genetic variation within the telomere-pathway gene loci examined may be useful predictor for T2D risk assessment.
 
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We compared cardiovascular disease outcomes according to the presence of peripheral arterial disease (PAD) at baseline in a post hoc analysis from the PROactive study. Of the 5238 patients in PROactive (a study of pioglitazone versus placebo in patients with type 2 diabetes and macrovascular disease; mean follow-up=34.5 months), 1274 had PAD at baseline (619=pioglitazone; 655=placebo). Patients with PAD at baseline showed significantly higher rates of the primary endpoint, main secondary endpoint, all-cause mortality (all P<0.0001), and stroke (P=0.0175) than those with no PAD at baseline. The risk of PAD alone was similar to that of myocardial infarction alone. In patients with no PAD at baseline, the event rates of the primary endpoint (P=0.0160), main secondary endpoint (P=0.0453), and acute coronary syndrome (P=0.0287) were significantly lower with pioglitazone than with placebo. This beneficial effect of pioglitazone was not seen in patients with PAD at baseline. In the total population, there was a higher frequency of leg revascularizations with pioglitazone than placebo-this was wholly due to first events that occurred within the initial 12 months of treatment. The presence of PAD increased the risk of all major cardiovascular events. Those without PAD at baseline seemed to benefit more from pioglitazone treatment than the overall PROactive population.
 
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To investigate the histopathologic modes of the effect of hypertension and hypercholesterolemia on atherosclerosis progression, a total of 573 male autopsied aortas, ranging from 0 to 97 years-old, were histomorphometrically compared by the status based on antemortem risk factors. Specimens were classified into four categories according to the criteria reported by the American Heart Association after histometric measurement at defined sites of the aortas. Intimal lesions progressed in the same fashion in all the risk factor groups examined; normal intima converted to fatty streak, preatheroma (characterized by microscopic extracellular lipid deposition) and then atheroma. This progression of intimal lesions correlated with age-related increases in intimal thickness independent of risk factors. Although the frequency of fatty streaks and the population of foam cells were greater in the hypercholesterolemics than in the non-risk patients, the frequencies of preatheroma and atheroma were not different between these two patient groups until patients reached the fifth decade. In contrast, the frequencies of preatheroma and atheroma were consistently greater in the hypertensives than in the other groups by the fifth decade. Hypertension was also related to intimal thickness in the younger groups. Our findings suggest that hypertension and hypercholesterolemia affect the progression of atherosclerosis differently by histopathologic stages.
 
Article
Lower activity of 11 beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2) classically induces hypertension by leading to an altered tetrahydrocortisol- versus tetrahydrocortisone-metabolites (THFs/THE) shuttle. Recent cell culture and animal studies suggest a role for promoter methylation, a major epigenetic feature of DNA, in regulation of HSD11B2 expression. Little is known, however, of human HSD11B2 epigenetic control and its relationship with the onset of hypertension. To explore the possible relevance of HSD11B2 promoter methylation, by examining human peripheral blood mononuclear cell (PBMC) DNA and urinary THFs/THE ratio as a biochemical indicator of 11beta-HSD2 activity, in blood pressure control. Twenty-five essential hypertensives and 32 subjects on prednisone therapy were analyzed, the latter to investigate 11beta-HSD2 function in the development of hypertension. Elevated HSD11B2 promoter methylation was associated with hypertension developing in glucocorticoid-treated patients in parallel with a higher urinary THFs/THE ratio. Essential hypertensives with elevated urinary THFs/THE ratio also showed higher HSD11B2 promoter methylation. These results show a clear link between the epigenetic regulation through repression of HSD11B2 in PBMC DNA and hypertension.
 
Article
The year 1997 celebrated the 20th anniversary of the European Lipoprotein Club. Sessions explored topics in the line of classical concepts and forthcoming advances in the field of basic and clinical research on lipoproteins. Participants from 18 European countries attended the conference. Recent Developments in Lipoprotein Research, were reviewed by Thomas Olivecrona (Umea, Sweden), who gave a perspective on lipolysis; and Gerd Assmann (Münster, Germany), who overviewed epidemiological data of the PROCAM study and focused on the biochemical and genetic components of reverse cholesterol transport. Session I, chaired by Katriina Aalto Setälä (Tampere, Finland) and Marten Hofker (Leiden, Netherlands) was dedicated to 'Lipoprotein receptors (old and new)'. Various structural and functional aspects were reported for the newcomers in the ever enriching LDL receptor gene family (VLDLR, LR7/8B, LR11, Megalin, RAP-related proteins). However, a decade of identification of LDL receptors gene defects reveals now that phenocopies of familial hypercholesterolemia may be linked to a third, yet unknown locus. Identification of pathways which clear HDL is underway. Session II, chaired by David Bowyer (Cambridge, United Kingdom) and Richard W James (Geneva, Switzerland), was entitled 'Significance of lipoprotein heterogeneity (metabolic and pathological aspects)'. Factors involved in lipoprotein modification (dense LDL, oxidation), transient production (post prandial, VLDL synthesis) or degradation (complement activation) and controversial hypotheses on their links with atherosclerosis were discussed. Session III on 'Novel methodologies for lipoprotein research' was chaired by Rudolph Poledne (Prague, Czech Republic) and Armin Steinmetz (Marburg, Germany). Simple technologies for routine assessment of lipoprotein metabolism, as well as the most sophisticated ones, to study lipid and free radical exchanges between particles, were presented.
 
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Guy De Backer
  • Ghent University
Michael Aviram
  • Technion - Israel Institute of Technology
Ian Graham
  • Trinity College Dublin
Jose M Ordovas
  • Tufts University
Ole Faergeman
  • Aarhus University