bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma membrane enrichment with cis-unsaturated fatty acids enhances LDL metabolism in U937 monocytes.
Authors: Kuo P , Weinfeld M , Rudd M A , Amarante P , Loscalzo J
Published: June 2011

The mechanism by which dietary cis-unsaturated fatty acids lower low density lipoprotein (LDL) cholesterol is unknown. Because cis-unsaturated fatty acids incorporated into cell membranes increase membrane fluidity and, as a result, can alter membrane-dependent cell functions, we examined LDL binding, uptake, and degradation in upregulated U937 monocytes enriched in membrane oleate, a monounsaturated fatty acid, and membrane linoleate, a polyunsaturated fatty acid. The same parameters were also examined in upregulated U937 monocytes enriched in membrane stearate, a saturated fatty acid, and in upregulated, unmodified U937 monocytes. Monocytes enriched in cis-unsaturated fatty acids exhibited augmented LDL binding, internalization, and degradation compared with both stearate-enriched monocytes and unmodified monocytes. The molar potency of linoleate in augmenting LDL metabolism was 50% greater than that of oleate. Enrichment with oleate and linoleate resulted in a decrease in the fatty acyl mole-weighted melting point of the plasma membrane and an increase in plasma membrane fluidity, as indicated by a reduction in the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into the membrane. Stearate-enriched monocytes exhibited a slight increase in the plasma membrane fatty acyl mole-weighted melting point and essentially no change in plasma membrane fluidity. Thus, one mechanism by which cis-unsaturated fatty acids lower LDL cholesterol may involve alteration in membrane lipid composition and physical properties, thereby leading to an increase in cellular clearance of this atherogenic lipoprotein.
Activation of endothelial cells induces platelet thrombus formation on their matrix. Studies of new in vitro thrombosis model with low molecular weight heparin as anticoagulant.
Authors: Zwaginga J J , Sixma J J , de Groot P G
Published: June 2011

Previous studies have indicated that activation of endothelial cells may lead to the production of tissue factor. We have studied the effect of endothelial cell activation and subsequent tissue factor synthesis on thrombus formation on the extracellular matrix in flowing blood. Endothelial cells were stimulated with tumor necrosis factor, endotoxin, or phorbol ester. Coverslips with activated cells or their extracellular matrix were introduced into a perfusion system and exposed to blood anticoagulated with 20 U/ml low molecular weight heparin. This concentration allowed manipulation of blood without activation of the coagulation cascade. Platelet deposition and fibrin formation were evaluated by morphometry, and fibrinopeptide A formation was assayed as a measure of thrombin generation. Activation of endothelial cells caused fibrinopeptide A generation in the perfusate and some deposition of fibrin on endothelial cells; however, platelets were not deposited. The matrix of the stimulated endothelium also caused enhanced fibrinopeptide A generation, and platelet aggregates and fibrin were deposited on the matrix. Maximal effects were observed with stimulation periods between 4 and 10 hours and were still clearly present after 18 hours. Increase in shear rate, perfusion time, and platelet number resulted in an increase in platelet adhesion, but platelet aggregate formation as a percentage of adhesion remained constant. Platelet aggregate formation and fibrinopeptide A generation were inhibited with antibodies against tissue factor or factor VIIa. Platelet aggregate formation alone was inhibited by antibodies against glycoprotein IIb/IIIa. Polymerization of fibrin on the matrix was best supported in perfusions at a low shear rate. The new in vitro thrombosis model presented here provides a powerful tool for study of the regulation of thrombogeneity by the vessel wall in response to various stimuli.
Persistent abnormalities in lipoprotein composition in noninsulin-dependent diabetes after intensive insulin therapy.
Authors: Bagdade J D , Buchanan W E , Kuusi T , Taskinen M R
Published: June 2011

To determine whether rigorous insulin therapy, which normalized the routinely measured plasma lipids, also reversed qualitative abnormalities in the composition of lipoproteins in noninsulin-dependent diabetes mellitus (NIDDM), we studied 18 NIDDM patients (eight men and 10 women) before and 2 months after intensive insulin therapy. Glycosylated hemoglobin levels (11.7% vs. 8.7%), plasma triglyceride (TG) (250 +/- 91 vs. 164 +/- 56 mg/dl, p less than 0.001), and cholesterol (214 +/- 43 vs. 198 +/- 31 mg/dl, p less than 0.025) all fell, and both HDL2 cholesterol and HDL3 cholesterol increased (59.1% and 10.9%, respectively, p less than 0.001). However, abnormalities in two indices of lipoprotein surface constituents, which were present before insulin therapy, remained so thereafter. The first of these, the new cardiovascular risk factor, the plasma free cholesterol/lecithin ratio, which was increased before treatment, fell only slightly after therapy (pre-therapy 1.02 +/- 0.29 vs. post-therapy 0.90 +/- 0.17, p less than 0.4; reference group, 0.83 +/- 0.14), and remained elevated in very low density lipoprotein (VLDL) and low density lipoprotein (LDL). Secondly, the sphingomyelin/lecithin ratio, an index of the surface rigidity of lipoproteins, was abnormal before treatment in VLDL, HDL2, and HDL3, and this alteration persisted after insulin therapy in HDL3 (p less than 0.001). Lipoprotein core lipid abnormalities were also present before treatment: the TG/cholesteryl ester ratio was reduced in VLDL and increased in LDL, HDL2, and HDL3. Rigorous insulin therapy improved, but failed to fully correct, this disturbance.(ABSTRACT TRUNCATED AT 250 WORDS)
Unexpected failure of bile acid malabsorption to stimulate cholesterol synthesis in sitosterolemia with xanthomatosis. Comparison with lovastatin.
Authors: Nguyen L , Salen G , Shefer S , Shore V , Tint G S , Ness G
Published: June 2011

We examined the relationship between cholesterol synthesis and high affinity low density lipoprotein (LDL) catabolism in freshly isolated mononuclear leukocytes and plasma sterols and apolipoprotein concentrations in three homozygous and one heterozygous subject with sitosterolemia with xanthomatosis and in 12 control subjects. Observations in untreated subjects were compared during therapy with lovastatin or interruption of the enterohepatic circulation of bile acids. Plasma cholesterol, plant sterol, and apolipoprotein B concentrations declined more than 50% in the two homozygous sitosterolemic subjects after ileal bypass surgery. In contrast, plasma cholesterol, plant sterol, and apolipoprotein B concentrations remained constant in a homozygous sitosterolemic subject and declined only 7% in a heterozygous sitosterolemic subject during 20 weeks of lovastatin (40 mg/day) treatment compared to a 28% decrease in similarly treated control subjects. Lovastatin treatment decreased cholesterol synthesis more than 60% but did not increase high affinity catabolism of LDL further in the sitosterolemic cells, compared to a more than 20% rise in control mononuclear leukocytes. Conversely, bile acid malabsorption increased cholesterol synthesis 59%, total hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activity 13%, and receptor-mediated LDL degradation 41% in control cells, but did not stimulate cholesterol synthesis or microsomal HMG-CoA reductase activity in sitosterolemic mononuclear leukocytes although receptor-mediated LDL catabolism rose an additional 26%. These results demonstrate a greater than expected decrease in plasma sterols and apolipoprotein B concentrations in sitosterolemic subjects after stimulation of bile acid synthesis because of the inability to up-regulate cholesterol production. We suggest that bile acid-sequestering drugs or ileal exclusion surgery may be more effective treatments to mobilize accumulated sterol deposits and prevent atherosclerosis in this disease.
Dynamic monitoring of platelet deposition on severely damaged vessel wall in flowing blood. Effects of different stenoses on thrombus growth.
Authors: Lassila R , Badimon J J , Vallabhajosula S , Badimon L
Published: June 2011

The formation of an arterial thrombus is a dynamic process that depends upon the characteristics of blood flow, the triggering substrate, and the blood components. We have developed and characterized a sensitive and specific computer-assisted nuclear scintigraphic method to study the dynamics of platelet deposition on severely damaged vessels both in vitro and in vivo in nonstenotic and stenotic flow conditions. Heparinized pig blood with Indium-111-labeled platelets was perfused for 50 minutes. Method variability in both static and flowing conditions was evaluated by Indium-111-labeled transferrin and Indium-111-labeled platelets. Positive scintigrams were obtained mainly in the presence of severe high grade stenoses on a thrombogenic substrate. Since the method is highly sensitive, computer-assisted axial dependence analysis was performed on the scintigraphic images to locate the thrombotic accumulation with respect to the area of the stenosis and to monitor the dynamic changes in platelet accumulation over time. Both in vitro and in vivo the highest level of platelet deposition occurred at the apex of the 80% stenosis, where embolization could be usually detected after 30 minutes of perfusion. This study is the first to assess the dynamics of thrombus growth in nonparallel flow streamlines such as are encountered in stenotic vessels. This method provides a new experimental tool with which to study factors affecting thrombus formation and stability.
Analysis of atherosclerosis susceptibility in mice with genetic defects in platelet function.
Authors: Paigen B , Holmes P A , Novak E K , Swank R T
Published: June 2011

To determine whether platelets contribute to the development of atherosclerosis, we compared the severity of atherosclerosis in susceptible C57BL/6 mice carrying either a normal or a variant phenotype for platelet function. Five genetically distinct mutants with increased bleeding times and abnormal dense granules were used: maroon (ru-2mr), light ear (le), ruby eye (ru), beige (bg1), and pale ear (ep). After a 14-week consumption of an atherogenic diet, three mutants had significantly less disease involvement than the control: light ear, maroon, and ruby eye. In contrast, pale ear ahd lesions similar to control animals. After 48 weeks, the two mutants with the least degree of atherosclerosis at 14 weeks, light ear and ruby eye, showed greater than 50% survival. In contrast, no animals from the beige, pale ear, or the normal C57BL/6 strains survived. To determine whether a specific biochemical component of platelet function is related to atherosclerosis, we measured serotonin found in dense granules. Serotonin showed no correlation with each mutant's atherosclerosis susceptibility. These results indicate that some particular component of platelet function affects atherosclerosis. That component is intact in pale ear, moderately affected in beige and maroon, and severely affected in light ear and ruby eye. The identity of that component remains an interesting question whose answer may provide further insight into the atherosclerotic disease process.
Effect of growth factors on human vascular endothelial cell prostacyclin production.
Authors: Ristimäki A , Ylikorkala O , Viinikka L
Published: June 2011

Prostacyclin (PGI2) is an antithrombotic factor, which may prevent the initiation and the complications of arteriosclerosis. The most important site of PGI2 production is the vascular endothelium, but little is known about how this process is regulated. In this connection, there is special interest in the roles of various growth factors released from platelets, macrophages, vascular smooth muscle cells, and the endothelial cells themselves. We investigated the effects of transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), and acidic and basic fibroblast growth factors (aFGF and bFGF) on the PGI2 production of cultured human umbilical vein endothelial cells by measuring the stable metabolite of PGI2, 6-keto-prostaglandin F1 alpha, by radioimmunoassay. TGF-beta induced dose- and time-dependent stimulation of PGI2 production. The lowest stimulatory concentration of TGF-beta was 0.1 ng/ml, and the maximal response, a 2.1-fold rise, was obtained with 1.0 ng/ml. The effect of TGF-beta lasted 48 hours and was blocked by inhibitors of transcription, translation, and cyclooxygenase. Maximal stimulation by TGF-beta was enhanced by epidermal growth factor. PDGF and bFGF had no effect on PGI2 production, but aFGF inhibited it. This is the first demonstration that TGF-beta enhances PGI2 production by human vascular cells, and this phenomenon may be part of negative feedback mechanisms that prevent thrombosis and arteriosclerosis.
Fibrinogen receptors do not undergo spontaneous redistribution on surface-activated platelets.
Authors: White J G , Escolar G
Published: June 2011

Studies with fibrinogen coupled to colloidal fibrinogen-gold (Fgn/Au) have suggested that fibrinogen receptors glycoprotein IIb-IIIa (GPIIb-IIIa) on human platelets may undergo spontaneous reorganization and centralization during surface activation. The present study has examined that hypothesis. Platelets were allowed to spread on grids for 20 minutes, just as in previous studies, but they were fixed before, rather than after, exposure to Fgn/Au or latex spherules. A monoclonal antibody to GPIIb-IIIa was also employed. Control experiments demonstrated that Fgn/Au and latex move toward the central zones and into channels of the open canalicular system on surface-activated platelets treated in the usual manner. However, when surface-activated platelets were fixed after spreading and before exposure to ligand, Fgn/Au particles and latex sperules were evenly dispersed over the entire cell membrane. Immunogold staining of GPIIb-IIIa also revealed edge-to-edge localization of the GPIIb-IIIa receptors on surface-activated cells. The findings are consistent with the concept that fibrinogen receptors do not undergo spontaneous reorganization on surface-activated platelets.
Relationship between high density lipoprotein subfractions and coronary risk factors in a rural white population.
Authors: Steenkamp H J , Jooste P L , Benadé A J , Langenhoven M L , Rossouw J E
Published: June 2011

The relationship of serum high density lipoprotein cholesterol (HDL-C) levels and of the HDL2-C and HDL3-C subfractions to several factors associated with coronary risk was examined in a cross-sectional study, which included 655 men and 731 women ages 20 to 64 years. Participants with coronary heart disease (CHD) had lower levels of HDL-C, HDL2-C, and HDL3-C; however, only HDL-C in women was significant. Maleness, body mass index, triglyceride levels, tobacco use, and carbohydrate intake (in men) were significantly inversely related to total HDL-C, while alcohol intake was significantly positively related to HDL-C. The associations were stronger for HDL2-C than for HDL3-C, except that alcohol intake in men was more strongly related to HDL3-C. The findings of this study suggest that several factors that influence CHD risk do so in part through modifying HDL2-C levels.
Whole blood impedance platelet aggregometry and ischemic heart disease. The Caerphilly Collaborative Heart Disease Study.
Authors: Elwood P C , Beswick A D , Sharp D S , Yarnell J W , Rogers S , Renaud S
Published: June 2011

The Caerphilly Collaborative Heart Disease Study is based on a large cohort of men who were ages 49 to 64 years at the time of the study. We report the results for platelet aggregation measured in whole blood from a subsample of 308 men. The index of sensitivity used was the minimum concentration of adenosine diphosphate that produced a defined degree of impedance change in the Chronolog 560 aggregometer. There was a marked association between aggregation and prevalent ischemic heart disease (IHD). The odds ratios and 95% confidence intervals (CI) for prevalent IHD in men with the most sensitive platelets compared with those with the least sensitive platelets were 3.6 (95% Cl: 1.1 to 12.2) for angina; 7.3 (95% Cl: 2.0 to 24.3) for previous myocardial infarction (MI); and 2.7 (95% Cl: 1.0 to 7.6) for electrocardiogram evidence of ischemia. The confidence limits for these odds ratios are large because of the small sample size, but the estimates of odds ratio are relatively large compared to similar relationships between the traditional risk factors of serum cholesterol, blood pressure, smoking, and prevalent IHD (1.5 to 2.5). A number of factors that might confound the relationships between platelets and IHD were examined, but the associations remained statistically significant when these were taken into account.
Abnormal composition of hypertriglyceridemic very low density lipoprotein determines abnormal cell metabolism.
Authors: Sehayek E , Eisenberg S
Published: June 2011

The metabolism of very low density lipoprotein (VLDL) from normolipemic (NTG) subjects, hypertriglyceridemic (HTG) subjects, and hypertriglyceridemic subjects treated with bezafibrate (BZ) was studied in cultured human skin fibroblasts. The binding, cell association, and proteolytic degradation of 125I-labeled lipoproteins and the capacity to regulate cellular sterol synthesis was determined with and without maximal stimulation of the lipoprotein by exogenous recombinant or plasmatic apolipoprotein (apo) E-3. The VLDL was separated into three density subfractions: I, II, and III. Multiple differences between HTG and NTG lipoproteins were found, which all reverted toward normal with therapy. Even in the presence of an optimal concentration of apo E-3, HTG-VLDL demonstrated 100% to 200% higher metabolic activities, indicating a better association or a better biological expression of apo E-3 at the surface of the lipoprotein. There was a strong and linear relationship between the cholesterol ester/protein ratios of the different VLDLs and their proteolytic degradations by the cells (r = 0.95). Thus, the composition/structure alterations of VLDL appear to determine their apo E-3-dependent cellular catabolism. In addition, HTG-VLDLs not enriched with apo E-3 exhibited a capacity to down-regulate cellular sterol synthesis independently of their uptake and degradation by the cells. This abnormality appeared to reflect the ability of the VLDL to donate cholesterol to the cells and was not observed in receptor-negative cells. Thus, HTG-VLDL is much more capable than NTG-VLDL of introducing cholesterol to cells by at least two mechanisms: 1) accelerated uptake and degradation and 2) direct transfer of cholesterol to the cells. Both processes are potentially atherogenic and are reversible when triglyceride-lowering therapy is instituted.
Vastatins inhibit cholesterol ester accumulation in human monocyte-derived macrophages.
Authors: Kempen H J , Vermeer M , de Wit E , Havekes L M
Published: June 2011

Human monocyte-derived macrophages were incubated for 48 hours in Medium 199 with 1% human serum albumin, and with 100 micrograms acetyl low density lipoprotein (LDL) or beta-very low density lipoprotein (beta-VLDL), with or without various concentrations of compactin, lovastatin, simvastatin, or pravastatin. The mass of free (FC) and esterified (CE) cholesterol was determined, as well as the incorporation of [1-14C]acetate in sterols, that of [1-14C]oleate in CE, and that of [methyl-14C]choline in phospholipids. Moreover, we assessed the high-affinity association and degradation of 125I-labeled acetyl LDL. Compactin markedly decreased the cellular accumulation of CE induced by acetyl LDL or beta-VLDL and increased the content of FC. Compactin also decreased the incorporation of [1-14C]oleate in CE (by 70-90%) in incubations with or without added lipoproteins. The half-maximal inhibitory concentration for this effect of compactin was 30 nM. Lovastatin and simvastatin were more potent, but pravastatin was about 100-fold less potent. Although compactin also caused a clear inhibition of cholesterol synthesis in the presence of acetyl LDL, the effect on CE formation did not seem to be related to decreased cholesterol synthesis, since this was already very low in the presence of acetyl LDL. Compactin did not affect the association and degradation of labeled acetyl LDL and also had no effect on the rate of cholesterol loss after preloading the cells with CE by incubation with acetyl LDL. However, compactin had a slight stimulatory effect on the synthesis of phosphatidylcholine and sphingomyelin when compactin was added to incubations in the presence of acetyl LDL.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of diet-induced hypercholesterolemia on proteoglycan metabolism in guinea pig megakaryocytes and platelets.
Authors: Schick B P
Published: June 2011

Proteoglycan metabolism was evaluated in megakaryocytes and platelets from guinea pigs fed a 1% cholesterol diet for 3 or 7 weeks. The animals were injected with a single dose of [35S]sulfate at the end of the feeding period, and megakaryocytes and platelets were isolated after 3 hours and then daily for 4 days thereafter. Proteoglycans were extracted from the cells of each animal and analyzed by ion-exchange chromatography, gel filtration, and electrophoresis. The maximal labeling of platelets occurred 2 days after [35S]sulfate injection as compared with 3 days in controls. A proteoglycan that eluted at Kav 0.2 from the Sepharose CL-6B column appeared 1 day after labeling. Additional proteoglycans of Kav 0.15 appeared at subsequent time points. The labeling profile for cholesterol-fed animals was unchanged from 2-4 days, unlike profiles from controls, which had exhibited a gradual increase in mean proteoglycan size. Thus, the progressive change in size of proteoglycans synthesized during normal megakaryocyte maturation was altered. The mean chain length of the proteoglycan-associated glycosaminoglycans from cholesterol-fed animals was increased relative to that of controls. In conjunction with the twofold increase in mean megakaryocyte size induced by cholesterol feeding in guinea pigs, the changes in proteoglycan synthesis suggest a state of stimulated megakaryocytopoiesis.
Effects of estrogen and progesterone on plasma lipoproteins and experimental atherosclerosis in the baboon (Papio sp.).
Authors: Kushwaha R S , Lewis D S , Carey K D , McGill H C
Published: June 2011

We determined the effect of estrogen and progesterone on plasma cholesterol and lipoprotein cholesterol concentrations and on arterial lesions in 24 ovariectomized and hysterectomized baboons fed a high-cholesterol/high-saturated-fat diet. These baboons were divided into four groups: untreated control (C); estrogen, 100 micrograms/kg/week injected i.m. (E); progesterone, 3 mg/kg/day (P); and estrogen plus progesterone (E + P). The treatment regimen continued for 18 months. Cholesterol levels in plasma and lipoproteins were measured before hormone treatment and at 3, 10, and 18 months of treatment. Postheparin plasma lipoprotein lipase (LPL) activity was also measured during the treatment. After 18 months of hormone treatment, baboons were necropsied and arterial lesions were measured. Hormone treatment significantly influenced plasma cholesterol (P greater than C greater than [E + P] greater than E) and very low density lipoprotein plus low density lipoprotein (VLDL + LDL) cholesterol (P greater than C greater than [E + P] greater than E), with very little effect on high density lipoprotein (HDL) cholesterol concentration. The E + P group had a significantly higher HDL cholesterol concentration than did the P group. The (VLDL + LDL)/HDL cholesterol ratios in the E and E + P groups were significantly lower than those in the P and C groups. LPL activities were significantly lower in the E group compared with those in the E + P and P groups. Hormone treatment significantly influenced lesions in four (innominate, carotid, iliac, and abdominal aorta) of seven arteries. The P group had the most fatty streaks, and the E + P group had the least.(ABSTRACT TRUNCATED AT 250 WORDS)
High fasting plasma insulin is an indicator of coronary heart disease in non-insulin-dependent diabetic patients and nondiabetic subjects.
Authors: Rönnemaa T , Laakso M , Pyörälä K , Kallio V , Puukka P
Published: June 2011

The association between fasting plasma insulin level and coronary heart disease (CHD) was studied in 909 non-insulin-dependent diabetic (NIDDM) patients, aged 45-64 years, and in 1,373 nondiabetic control subjects. Both diabetic and nondiabetic subjects with various manifestations of CHD had higher plasma insulin levels than did subjects free of CHD. By plasma insulin quintiles formed according to values in nondiabetic subjects, the age-adjusted prevalence of CHD defined by symptoms and/or electrocardiographic changes in diabetic men was 48.2% in quintiles I + II (lowest), 54.8% in quintiles III + IV, and 65.7% in quintile V (highest) (p = 0.006). The respective prevalences in diabetic women were 53.5%, 59.1%, and 73.3% (p = 0.004); in nondiabetic men, 28.1%, 33.7%, and 43.3%, respectively (p = 0.016); and in nondiabetic women, 28.1%, 34.9%, and 44.3%, respectively (p = 0.007). An essentially similar association was observed between plasma insulin level and definite or possible myocardial infarction (MI). In diabetic subjects, a positive association between plasma insulin level and CHD manifestations was also found when insulin strata were formed using quintile cutoff points determined separately from diabetic subjects. The association between plasma insulin level and the prevalence of CHD or MI disappeared or was weaker, especially in men, when adjustment was made for body mass index, hypertension, and triglyceride or high density lipoprotein (HDL) cholesterol level. The association between high plasma insulin level and CHD was significant in diabetic subjects with a body mass index greater than 27 kg/m2 but not in those diabetics with a body mass index less than or equal to 27 kg/m2. A significant clustering of hypertension, high triglyceride values, and low HDL cholesterol levels was observed in diabetic subjects in the highest insulin quintiles. The results suggest that hyperinsulinemia is an indicator of CHD in both NIDDM patients and nondiabetic subjects. Hyperinsulinemia may be directly atherogenic, but it is more probable that hyperinsulinemia reflects insulin resistance, which may be a factor enhancing atherogenesis by causing adverse changes in many CHD risk factors.
Cholesterol accumulation in J774 macrophages induced by triglyceride-rich lipoproteins. Comparison of very low density lipoprotein from subjects with type III, IV, and V hyperlipoproteinemias.
Authors: Huff M W , Evans A J , Sawyez C G , Wolfe B M , Nestel P J
Published: June 2011

The capacity of human triglyceride-rich lipoproteins to induce cholesterol accumulation in the murine J774 macrophage cell line was investigated with large very low density lipoprotein (VLDL, Sf 60-400) obtained from subjects with type III, IV, and V hyperlipoproteinemias. After incubation for 24 hours, VLDLs from type IV and type V subjects were similar in their ability to raise cellular cholesterol deposition threefold to fourfold and cellular triglyceride 16-fold. The increase in cholesterol was entirely due to the dramatic increase in cholesterol ester, from less than 1 to greater than 50 micrograms/mg cell protein. Total cholesterol accumulation was fourfold to fivefold greater than the cholesterol accumulation observed for VLDL or low density lipoprotein (LDL) from normal subjects. Cholesterol esterification (acyl coenzyme A: cholesterol acyltransferase [ACAT] activity) paralleled the rate of cholesterol accumulation in these cells. Treating the macrophages with the ACAT inhibitor 58035, which is known to downregulate the LDL receptor in these cells, diminished cholesterol accumulation by 40% for type IV VLDL and by 23% for normal LDL. Since hypertriglyceridemic VLDL carries excess apoprotein (apo) E molecules, we investigated the role of normal and abnormal apo E. An anti-apo E monoclonal antibody, known to block the binding of apo E to the LDL receptor, blocked type IV VLDL-induced cholesterol ester accumulation by approximately 70%. In contrast to type IV subjects, VLDL from type III subjects (homozygous for apo E2) when incubated with J774 macrophages (which do not secrete apo E) caused only a modest 1.5-2-fold increase in cellular cholesterol. Pre-beta- and beta-migrating VLDL subfractions from type III subjects were equally ineffective in causing cholesterol accumulation. By contrast, beta-VLDL from cholesterol-fed rabbits caused a sevenfold to eightfold increase in cellular cholesterol content. These results indicate that triglyceride-rich lipoproteins from type IV and type V subjects can cause substantial cholesterol ester accumulation and enhanced cholesterol esterification in J774 cells. The lower cholesterol accumulation with type IV VLDL in the presence of apo E antibodies and VLDL from type III subjects demonstrates the importance of functional apo E in this process.
Display of low density lipoprotein receptors is clustered, not dispersed, in fibroblast and hepatocyte plasma membranes.
Authors: Robenek H , Harrach B , Severs N J
Published: June 2011

Although the principal details of low density lipoprotein (LDL) uptake by receptor-mediated endocytosis and its subsequent intracellular fate have been thoroughly investigated, an aspect of this mechanism that continues to provoke controversy concerns the manner of display of LDL receptors upon their initial insertion at the cell surface. While our studies based on electron microscopy of platinum/carbon replicas of gold-labeled cells have previously suggested a clustered display pattern, others have concluded, before and since, that LDL receptors are inserted individually at random widely dispersed sites in the plasma membrane. In this article, we present a series of experiments designed to discriminate between these competing hypotheses. In addition to the use of LDL-colloidal gold complexes, visualized electron microscopically, on cells subjected to a variety of experimental procedures, these experiments include the application of anti-apolipoprotein B-100 antibodies, anti-LDL-receptor antibodies, and direct visualization of native (unlabeled) LDL molecules at the cell surface. All results point to a loose-cluster arrangement, not one involving widely dispersed individual units, as the initial display pattern of newly inserted LDL receptors. A comparison of LDL and beta-very low density lipoprotein receptor distribution in fibroblasts and hepatocytes suggests that this cluster pattern is a characteristic of the LDL (apolipoprotein B/E) receptor across cell types, but that the closely related apolipoprotein E receptor differs in that it is inserted individually in a highly dispersed state, in common with a variety of other receptor types.
Relation of serum testosterone levels to high density lipoprotein cholesterol and other characteristics in men.
Authors: Freedman D S , O'Brien T R , Flanders W D , DeStefano F , Barboriak J J
Published: June 2011

Although levels of high density lipoprotein (HDL) cholesterol in males decrease during adolescence and after treatment with testosterone derivatives, several studies have reported that levels of HDL cholesterol are positively associated with endogenous levels of testosterone in men. This association was further examined using data collected during 1985 and 1986 from 3,562 white and 500 black men who ranged in age from 31 to 45 years. Black men had higher mean levels of both HDL cholesterol (8 mg/dl) and total testosterone (33 ng/dl) than white men, and positive associations were observed between testosterone and HDL cholesterol levels (r = 0.22, whites; r = 0.26, blacks). In addition, levels of testosterone were related positively to alcohol consumption and cigarette smoking and negatively to age, Quetelet index, and use of beta-blockers. We used stratification and regression analyses to determine if any of these characteristics could account for the positive association between levels of HDL cholesterol and total testosterone. Although controlling for most factors had little influence, adjusting for Quetelet index reduced the strength of the association between levels of testosterone and HDL cholesterol by approximately 30%. These findings suggest that the positive association between levels of testosterone and HDL cholesterol may not be causal. Multivariable analyses that control for obesity and other potentially confounding characteristics should be used in studies that assess the relation of testosterone levels to coronary heart disease.
Central adipose distribution is related to coronary atherosclerosis.
Authors: Thompson C J , Ryu J E , Craven T E , Kahl F R , Crouse J R
Published: June 2011

A "male" distribution of adipose tissue in women (excess of fat in the abdomen compared with that in the hips; i.e., elevated waist/hip ratio) has been related to symptomatic cardiovascular disease. An elevated waist/hip ratio has also been related to symptomatic cardiovascular and cerebrovascular diseases in men, as well as to risk factors for these diseases and various metabolic conditions. To determine whether adipose distribution was related to coronary atherosclerosis, we performed a case-control study in patients with angiographically documented coronary atherosclerosis (cases) and in angiographically normal hospital and neighborhood controls. The data show that distribution of adiposity as assessed by waist/hip ratio is significantly related to coronary atherosclerosis in both females and males. Waist/hip ratio is significantly greater in female cases compared with either control group; in males, waist/hip ratio is significantly greater in cases compared with asymptomatic neighborhood controls but not compared with patients with normal coronary arteries. These results persist after control for age, plasma concentrations of lipids and lipoproteins, body mass index, history of hypertension, history of diabetes, and smoking status. The connection between the male adipose distribution in females and coronary atherosclerosis partly explains the greater likelihood of symptomatic cardiovascular disease in them. Males with excess deposition of fat in the abdominal region are also likely to experience increased risk.
Apolipoprotein B gene mutations in Austrian subjects with heart disease and their kindred.
Authors: Friedl W , Ludwig E H , Balestra M E , Arnold K S , Paulweber B , Sandhofer F , McCarthy B J , Innerarity T L
Published: June 2011

In a group of 110 subjects with severe coronary artery disease, two were heterozygous for the apolipoprotein (apo) B arginine3,500----glutamine mutation that characterizes familial defective apo B-100. Both affected subjects were moderately hypercholesterolemic, and their low density lipoproteins (LDLs) were deficient in binding to the LDL receptor. Pedigree analysis of the two probands' families established a correlation between the apo B mutation, defective LDL, and a particular apo B haplotype that was characterized by 10 apo B gene markers. In addition to having one allele carrying the arginine3,500----glutamine mutation, one family member may harbor a second mutant apo B allele that causes its gene product to be present in plasma at a lower than normal level, despite the fact that the affinity of the protein for the LDL receptor appears to be normal. The metabolic basis for the underrepresentation of this second allotype remains to be elucidated.
Effect of interleukin-1 on lipid metabolism in the rat. Similarities to and differences from tumor necrosis factor.
Authors: Feingold K R , Soued M , Adi S , Staprans I , Neese R , Shigenaga J , Doerrler W , Moser A , Dinarello C A , Grunfeld C
Published: June 2011

Infection and inflammation are associated with hypertriglyceridemia, which is thought to be mediated by cytokines. Previous studies at our laboratory and others have shown that tumor necrosis factor acutely increases serum triglyceride levels primarily by stimulating hepatic lipid synthesis and secretion. The role of interleukin-1 (IL-1), a cytokine that is also secreted by stimulated macrophages and that has many actions that overlap those of tumor necrosis factor, has not been studied in depth. The present study demonstrates that IL-1, at doses similar to those that cause fever and anorexia and that stimulate adrenocorticotropic hormone secretion, rapidly increases serum triglyceride levels; this elevation persists for at least 17 hours. Serum cholesterol levels are not altered by IL-1. Neither is the clearance of triglyceride-rich lipoproteins affected by IL-1. However, hepatic triglyceride secretion, measured by the Triton WR-1339 technique, is increased in IL-1-treated animals. Accompanying this stimulation in hepatic lipid secretion is an increase in de novo fatty acid synthesis in the liver. IL-1 does not increase serum free fatty acid and glycerol levels, suggesting that IL-1 does not stimulate lipolysis in vivo. Additionally, inhibition of lipolysis does not prevent the increase in serum triglyceride levels, providing further evidence that lipolysis does not play a crucial role in the increased hepatic lipid synthesis and secretion induced by IL-1. In contrast, tumor necrosis factor increases lipolysis, which contributes to the increase in serum triglycerides. That multiple cytokines rapidly elevate plasma triglyceride levels suggest that these changes in lipid metabolism may play an important role in the organism's response to infection and inflammation.
Intimal alterations in rabbit aortas during the first 6 months of alloxan-induced diabetes.
Authors: Hadcock S , Richardson M , Winocour P D , Hatton M W
Published: June 2011

Diabetes mellitus is a major risk factor for atherosclerosis. Since endothelial alteration is probably associated with the development of atherosclerosis, we questioned whether morphological evidence of endothelial injury could be observed during the first 6 months of diabetes induced by a single intravenous injection of alloxan in normally fed rabbits compared with age-matched controls. Diabetes (plasma glucose greater than 16 mM) was established by 5 days after alloxan injection. Endothelial alterations consistent with injury, including adhesion of white blood cells, platelets, and fibrin-like material to the endothelial surface, were seen in diabetic rabbit aortas by 2 weeks. These alterations became more severe during the next 6 months. Increased endothelial replication in diabetic vessels was shown by the uptake of tritium-labeled thymidine at 2 weeks and at 3 and 6 months. Hyperplasia of intimal smooth muscle cells progressed during 3 months after treatment. About one third of the diabetic rabbits also showed an elevated plasma cholesterol level, which correlated with increased intimal proliferation but not with endothelial injury or replication. The onset of alloxan-induced diabetes in rabbits is associated with nondenuding endothelial injury and subsequent intimal hypertrophy, changes that are consistent with atherogenesis.
Antithrombin III-beta associates more readily than antithrombin III-alpha with uninjured and de-endothelialized aortic wall in vitro and in vivo.
Authors: Witmer M R , Hatton M W
Published: June 2011

The properties of two isoforms, alpha and beta, of rabbit antithrombin III (ATIII) were compared in the presence of undamaged or de-endothelialized rabbit aortic wall. Similar quantities of ATIII-alpha and ATIII-beta bound to and rapidly saturated the endothelium in vitro, but the rate of transendothelial passage of ATIII-beta exceeded that of ATIII-alpha by 22%. Furthermore, ATIII-beta was adsorbed approximately twice as rapidly as ATIII-alpha by the subendothelium of the de-endothelialized aorta. Binding of both isoforms was decreased (ATIII-beta more than ATIII-alpha) by pretreating the subendothelial surface with heparitinase. Also, subendothelium-bound ATIII-beta was desorbed more readily than bound ATIII-alpha by thrombin. In vivo, the rate of uptake of iodine-131-labeled ATIII-beta from the circulation by the aortic wall and the major organs was 30-50% faster than that of iodine-125-labeled ATIII-alpha. In contrast, the uptake of 131I-ATIII-beta by the de-endothelialized aorta in vivo was three times faster than that of 125I-ATIII-alpha. By these criteria, ATIII-beta is the more active of the two isoforms. We surmise that plasma and, consequently, vessel wall levels of ATIII-beta may be vital for controlling thrombogenic events caused by injury to the vascular wall.
Inhibition of platelet retention on artificial microvascular grafts with monoclonal antibodies and a high-affinity peptide directed against platelet membrane glycoproteins.
Authors: Johnson P C , Sheppeck R A , Hribar S R , Bentz M L , Janosky J , Dickson C S
Published: June 2011

Rapid occlusion of artificial microvascular grafts (AMGs; less than or equal to 2-mm diameter) by platelet-rich thrombi prevents the clinical use of AMGs in cardiac, vascular, and plastic surgery. Since present antiplatelet agents are unable to assure AMG patency, we have studied the role of specific platelet membrane-glycoprotein blockade on platelet retention by AMGs. In a customized in vitro perfusion chamber, retention on polytetrafluoroethylene (PTFE) AMGs (1.0-mm i.d.) of indium-111-labeled platelets in human whole blood was measured in the presence and absence of inhibitors. Specific blockade of platelet membrane glycoprotein (Gp) IIb/IIIa was achieved using monoclonal antibody 10E5 (10 micrograms/ml) and the peptide GRGDS (Gly-Arg-Gly-Asp-Ser, 0.75 mM). These inhibited 98% and 35%, respectively, of platelet retention under circumstances in which aspirin (7 mM) and dextran (4 mg/ml) inhibited 19% and 18%, respectively, of platelet retention. Nonspecific immunoglobulin G F(ab')2 (10 micrograms/ml) and nonspecific peptide (GGDA; Gly-Gly-Asp-Ala, 0.75 mM), used as control reagents, were ineffective in this setting. Monoclonal antibody 6D1 (10 micrograms/ml), which blocks platelet membrane GpIb, prevented 82% of platelet retention on PTFE. These doses of 10E5 and GRGDS completely inhibited platelet aggregation in response to 20 microM ADP, and the dose of 6D1 completely inhibited ristocetin-induced platelet agglutination. The aspirin dose prevented the second phase of ADP-induced aggregation. These data indicate that not only does initial platelet adhesion to PTFE require GpIIb/IIIa but also that GpIb plays a significant role in the early stages of platelet retention on PTFE AMGs.
Quantitative studies of transfer in vivo of low density, Sf 12-60, and Sf 60-400 lipoproteins between plasma and arterial intima in humans.
Authors: Shaikh M , Wootton R , Nordestgaard B G , Baskerville P , Lumley J S , La Ville A E , Quiney J , Lewis B
Published: June 2011

To assess the potential of various plasma lipoprotein classes to contribute to the lipid content of the arterial intima, influx and efflux of these plasma lipoprotein fractions into and from the intima of human carotid arteries were measured in vivo. While low density lipoprotein (LDL) is known to transfer from plasma into the arterial wall, there is less information on the atherogenic potential of lipoproteins of intermediate density (Sf 12-60) or of very low density (Sf 60-400). Aliquots of the same lipoprotein (LDL, Sf 12-60 lipoprotein particles, or Sf 60-400 lipoprotein particles) iodinated with iodine-125 and iodine-131 were injected intravenously 18-29 hours and 3-6 hours, respectively, before elective surgical removal of atheromatous arterial tissue, and the intimal clearance of lipoproteins, lipoprotein influx, and fractional loss of newly entered lipoproteins were calculated. Intimal clearance of Sf 60-400 particles was not detectable (less than 0.3 microliter x hr-1 x cm-2), whereas the average value for both LDL and Sf 12-60 lipoprotein particles was 0.9 microliter x hr-1 x cm-2. Since the fractional loss of newly entered LDL and Sf 12-60 lipoprotein particles was also similar, the results suggest similar modes of entry and exit for these two particles. However, due to lower plasma concentrations of Sf 12-60 lipoproteins as compared with LDL, the mass influx of cholesterol in the Sf 12-60 particles was on the order of one 10th of that in LDL, and that of apolipoprotein B was about one 20th.(ABSTRACT TRUNCATED AT 250 WORDS)
Alterations in human vascular endothelial cell function by oxygen free radicals. Platelet adherence and prostacyclin release.
Authors: Shatos M A , Doherty J M , Hoak J C
Published: June 2011

This study was directed to the ability of oxygen free radicals to cause reversible vascular endothelial cell dysfunction. A well-characterized system for the production of the superoxide anion radical (O2(-).) and hydrogen peroxide (H2O2), employing xanthine and xanthine oxidase, was used to sublethally injure human umbilical vein endothelial (HUVE) cells in vitro. We examined the effects of a 15-minute incubation of HUVE cells with xanthine (50 microM) and xanthine oxidase (2.5-100 munits) on platelet adherence and prostacyclin (PGI2) release. All experiments were conducted in a serum-free N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)-Tyrode buffer (pH 7.4) incubation system. Exposure of HUVE cells to sublethal concentrations of oxygen free radicals caused significant enhancement of platelet adherence (65 +/- 6.3%) to injured endothelium. A 12-fold increase in PGI2 release resulted after a 15-minute treatment with xanthine and xanthine oxidase. The addition of exogenous PGI2 (150 mM) to platelet-endothelial systems did not completely prevent the enhanced platelet adherence, suggesting that a lack of PGI2 was not completely responsible for the adherence of platelets to O2(-).-injured cells. When superoxide dismutase (SOD) and catalase, scavengers of O2(-). and H2O2, were added in combination to treated cells, platelet adherence decreased by 42-77% and PGI2 release approached that of control cultures. No decrease in either platelet adherence or PGI2 release occurred when chemically inactivated forms of SOD and catalase or bovine serum albumin were added to oxidant-treated cultures.
Long-term cholesterol feeding alters the reactivity of primate coronary microvessels to platelet products.
Authors: Quillen J E , Sellke F W , Armstrong M L , Harrison D G
Published: June 2011

Hypercholesterolemia impairs endothelium-dependent relaxations to several platelet-derived substances in large vessels. The effect of hypercholesterolemia on the response of coronary microvessels to platelet products and thrombin is less well defined. Three groups of cynomolgus monkeys were studied: normal (n = 6), short-term hypercholesterolemic (8-11 weeks, n = 5), and long-term hypercholesterolemic (18-80 months, n = 6). Responses of coronary microvessels, 100-200 microns in diameter, to thrombin (0.1-10 units/ml) and the platelet products ADP (1 nM-100 microM), serotonin (1 nM-100 microM), and the thromboxane A2 analogue U46619 were studied using an in vitro microvessel imaging apparatus. Vessels were studied after preconstriction with thromboxane A2 analogue U46619 to evaluate both relaxations and constrictions to each agent. Concentrations of U46619 to attain preconstriction were much lower for the long-term group (16 +/- 19 nM) as compared with the control and short-term hypercholesterolemic groups (689 +/- 48 and 664 +/- 63 nM, respectively, p less than 0.01). Relaxations of long-term hypercholesterolemic vessels to ADP tended to be less than those of either control or short-term hypercholesterolemic vessels. Thrombin, when added to normal or short-term hypercholesterolemic vessels, caused identical relaxations but paradoxically caused constrictions in microvessels of long-term hypercholesterolemic monkeys (55 +/- 17% of KCl contraction, p less than 0.001 vs. other groups). Peak constrictions to serotonin were markedly enhanced in the long-term hypercholesterolemic group (59 +/- 7% of maximal KCl responses) compared with control and short-term hypercholesterolemic responses (28 +/- 8% and 32 +/- 5%, respectively, both p less than 0.05 vs. long-term hypercholesterolemia.(ABSTRACT TRUNCATED AT 250 WORDS)