Lipoprotein-acid mucopolysaccharide complexes of human atherosclerotic lesions.

Lipoprotein-acid mucopolysaccharide complexes occurring in different types of human atherosclerotic lesions were isolated and partially characterized. Complexes from fatty streaks and fibrous plaques were extracted from pooled tissues with 0.15 M NaCl, purified by gel filtration, and fractionated by ultracentrifugation. Both low and very low density lipoproteins were present; low density lipoprotein was predominant. The complexes were analyzed for uronic acid, cholesterol, phospholipid, and Ca-2+ contents; there was no significant difference in the relative molar ratios between complexes from fatty streaks and fibrous plaques. Acid mucopolysaccharides were isolated from the complexes and identified by electrophoresis and enzymatic studies. Chondroitin sulfate C and hyaluronic acid were found in complexes from fatty streaks and fibrous plaques. Heparin was detected only in fibrous plaques.     

Influence of dietary trans-fatty acids on swine lipoprotein composition and structure.

Four groups of 20 weanling swine each were fed either (a) basal diet, (b) basal plus hydrogenated fat (13% trans), (c) basal plus hydrogenated fat (13% trans) and 0.4% cholesterol, or (d) basal plus beef tallow (all cis). After six months of feeding, the animals were killed and the blood and aortas were removed. Very low density, low density, and high density lipoproteins were then isolated from the plasma by ultracentrifugal flotation. Although the fatty acid composition of the basal diet was different from the diets supplemented with either hydrogenated fat containing trans-fatty acid or beef tallow containing all cis, the lipid and fatty acid compositions of each of the isolated lipoprotein classes for the four groups of animals were remarkably similar. Elaidate was clearly incorporated into the lipoproteins of animals fed hydrogenated fat, but the level of incorporation was generally less than 5%. In a direct comparison of the structure of the lipoproteins from the different groups, we did not find any significant differences in their physical properties as determined by pyrene fluorescence and electron paramagnetic resonance methods. Grossly visible fatty streaks and fibrous plaques were not found in any of the swine aorta. However, light and electron microscopy indicated the presence of atherosclerotic lesions in the distal abdominal aorta and bifurcation. These studies demonstrate that a diet containing a substantial amount of trans-fatty acid leads to a small but definite incorporation into the swine lipoproteins. However, such changes had relatively little effect on lipoprotein structure or the presence of atherosclerotic lesions in these 6-month-old swine.     

Oxysterol profiles of normal human arteries, fatty streaks and advanced lesions.

OBJECTIVE: Human atherosclerotic lesions of different stages have quantitative differences in cholesterol and oxysterol content, but information on the oxysterol profile in fatty streaks is limited. This study aims to provide more detailed oxysterol quantification in human fatty streaks, as well as normal aorta and advanced lesions. METHODS: A newly adapted method was used, including oxysterol purification by means of a silica cartridge; and it was ensured that artifactual oxysterol formation was kept to a minimum. Cholesterol and oxysterols were estimated by GC and identification confirmed by GC-MS in samples of normal human arterial intima, intima with near-confluent fatty streaks and advanced lesions, in necropsy samples. RESULTS: The oxysterols 7 alpha-hydroxycholesterol, cholesterol-5 beta, 6 beta-epoxide, cholesterol-5 alpha, 6 alpha-epoxide, 7 beta-hydroxycholesterol, 7-ketocholesterol and 27-hydroxycholesterol (formerly known as 26-hydroxycholesterol) were found in all the lesions, but were at most very low in the normal aorta, both when related to wet weight and when related to cholesterol. Most components of the normal artery showed some cross-correlation on linear regression analysis, but cross-correlations were weaker in the fatty streaks and advanced lesions. However, in fatty streak there was a marked positive correlation between 27-hydroxycholesterol and cholesterol. CONCLUSION: The findings confirm that oxysterols are present in fatty streaks and advanced lesions and may arise from different cholesterol oxidation mechanisms, including free radical-mediated oxidation and enzymatic oxidation.     

Role of heparanase on hepatic uptake of intestinal derived lipoprotein and fatty streak formation in mice

Background

Heparanase modulates the level of heparan sulfate proteoglycans (HSPGs) which have an important role in multiple cellular processes. Recent studies indicate that HSPGs have an important function in hepatic lipoprotein handling and processes involving removal of lipoprotein particles.

Principal Findings

To determine the effects of decreased HSPGs chain length on lipoprotein metabolism and atherosclerosis, transgenic mice over-expressing the human heparanase gene were studied.Hepatic lipid uptake in hpa-Tg mice were evaluated by giving transgenic mice oral fat loads and labeled retinol. Sections of aorta from mice over-expressing heparanase (hpa-Tg) and controls (C57/BL6) fed an atherogenic diet were examined for evidence of atherosclerosis. Heparanase over-expression results in reduced hepatic clearance of postprandial lipoproteins and higher levels of fasting and postprandial serum triglycerides. Heparanase over-expression also induces formation of fatty streaks in the aorta. The mean lesion cross-sectional area in heparanase over-expressing mice was almost 6 times higher when compared to control mice (23,984 µm²±5,922 vs. 4,189 µm²±1,130, p<0.001).

Conclusions

Over-expression of heparanase demonstrates the importance of HSPGs for the uptake of intestinal derived lipoproteins and its role in the formation of fatty streaks.     

Effect of phospholipase A treatment of low density lipoproteins on the dextran sulfate--lipoprotein interaction

The effect of phospholipase A on the interaction of low density lipoproteins of the S(f) 0-10 class with dextran sulfate was studied in phosphate buffer of pH 7.4, ionic strength 0.1, by chemical, spectrophotometric, and centrifugal methods. When low density lipoproteins that had been treated with phospholipase A were substituted for untreated lipoproteins, the amount of insoluble dextran sulfate-lipoprotein complex formed was greatly reduced. Hydrolysis of over 20% of the lecithin and phosphatidyl ethanolamine constituents of the lipoproteins prevented the formation of insoluble complex. However, even the lipoproteins in which almost all the phosphoglycerides were hydrolyzed produced soluble complex, which was converted to insoluble complex upon addition of magnesium sulfate. It is apparent that the lipoproteins altered extensively by treatment with phospholipase A retain many characteristic properties of native low density lipoproteins. Fatty acids, but not lysolecithin, released by the action of phospholipase A interfered with the formation of insoluble complex; this interference was due to association of the fatty acids with the lipoproteins. With increases in the concentration of the associated fatty acids, the amounts of magnesium ion required for the conversion of soluble complex to insoluble complex increased progressively. Charge interaction is evidently of paramount importance in the formation of sulfated polysaccharide-lipoprotein complexes.     

Action of leptospiral lipases on purified serum lipoproteins

Partially purified exocellular lipases produced by a pathogenic (Leptospira copenhageni (PB-3) and a saprophytic strain ofLeptospirae (Leptospira Patoc I) hydrolyzed very low density, and to a lesser extent low density hog serum lipoproteins. No high density lipoprotein hydrolysis was found. Optimal conditions for the action of both lipases on very low density serum lipoproteins were determined: pH 8.5; NaCl 0.4m; CaCl₂ 1.0mm; sodium desoxycholate 10.60mm. The maximal velocities of very low density lipoprotein hydrolysis were compared with the maximal velocities of triglyceride emulsion hydrolysis by both lipases.     

Differential influence of lipoproteins isolated from women and men on the activity of the PGI2 synthetase activity

We have found a correlation between the activity of the PGI₂ synthetase in the microsomal fraction of pig aorta and the amount of high density lipoprotein (HDL) cholesterol in the incubation fluid. The reverse was true for low density lipoprotein (LDL) cholesterol. These correlations exist independently of whether the lipoproteins were isolated from men than from women as it is for HDL isolated from women than from men. This result may give an explanation for the differential risk against the incidence of cardiovascular diseases between women and men at the same concentration of the individual lipoproteins in their blood.     

人动脉壁蛋白聚糖与血清脂蛋白形成的不溶性复合物

本文报道了人主动脉壁中正常及异常(脂纹,FS)区的三种蛋白聚糖(PG)即:硫酸软骨素PG(CSPG)、硫酸皮肤素—硫酸软骨素PG(DSCSPG)及硫酸乙酰肝素PG(HSPG)与血清极低密度脂蛋白(VLDL)及低密度脂蛋白(LDL)所形成的不溶性复合物。在30mmol/L Ca~(2+)对,三种PG都能与这两种脂蛋白形成不溶性复合物,随放置时间的增加,形成的复合物都发生解离,但其复合物形成的曲线及解离程度明显不同。DSCSPG与CSPG比较,前者与两种脂蛋白更易形成不溶性复合物且不易解离。HSPG与两种脂蛋白形成不溶性复合物所需时间远大于CSPG及DSCSPG。FS区及正常区三种PG形成复合物曲线类型相似,异常区CSPG、DSCSPG与VLDL形成的复合物量低于正常区的相应PG,而与VLDL则高于正常区的相应PG。异常区的HSPG与两种脂蛋白形成不溶性复合物的量均高于正常区。     

Increased susceptibility to peroxidation of VLDL from non-insulin-dependent diabetic patients: A possible correlation with fatty acid composition

Recent studies suggested that both oxidized very low density lipoproteins (VLDL) and oxidized high density lipoproteins (HDL) might play a role in the pathogenesis of atherosclerosis. The aim of the present work was to analyse the susceptibility to in vitro peroxidation of VLDL and HDL from apparently normolipidemic subjects affected by insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) in good metabolic control and to examine the possible relations between oxidisability and lipoprotein fatty acid composition. VLDL and HDL were isolated from 13 IDDM patients, 12 NIDDM patients and 18 healthy subjects. The degree of lipoprotein oxidation was determined by the measurement of hydroperoxide levels and thiobarbituric acid-reactive substances (TBARS) before and after in vitro peroxidative stress with CuSO₄. Fatty acid analysis was performed by gas chromatography. VLDL and HDL from NIDDM patients showed a decrease in the saturated fatty acid content with a concomitant increase in unsaturated fatty acids and higher basal peroxide levels compared with healthy subjects. Oxidisability of VLDL from NIDDM subjects was higher than in controls and was significantly related with the unsaturated fatty acid content. The present work suggests that alterations in the composition and functions of both VLDL and HDL able to produce more atherogenic lipoproteins are present in NIDDM.     

Regression of diet-induced atherosclerosis in G?ttingen miniature swine

The regression of diet-induced atherosclerosis in G?ttingen Miniature Swine was investigated after a 6-month induction period. At 1 month after feeding a high-cholesterol and high-fat diet, levels of beta-lipoprotein, total cholesterol, free fatty acids and phospholipid had increased rapidly and the high levels were maintained throughout the 6 month induction period. Morphological features at 6 months showed fatty streaks in the thoracic aorta and fibrous plaques in the abdominal aorta. After return to the conventional diet at 6 months, serum lipids decreased rapidly and maintained the baseline level throughout the 9 month regression period. Histopathological findings showed the regression of fatty streaks but the fibrous plaques did not regress. The present study therefore confirms the regression of fatty streaks in the aorta of G?ttingen Miniature Swine by the administration of a cholesterol lowering diet.     

Regulation of lipogenesis in isolated hepatocytes by triglyceride-rich lipoproteins.

Very low density lipoproteins, chylomicrons, and remnants caused, within an hour, significant inhibition of fatty acid synthesis but not cholesterol synthesis in hepatocytes isolated from meal-fed rats. In contrast, low density lipoproteins, high density lipoproteins, and the serum fraction of density greater than 1.21 failed to significantly inhibit either fatty acid or cholesterol synthesis within 1 h. The Scatchard plots of specific binding showed that rat and human very low density lipoproteins interact with the high affinity sites on the hepatocytes with the apparent dissociation constants of 64 and 106 nM, respectively. These data also indicated that each hepatocyte was capable of binding 6 X 10(5) molecules of very low density lipoproteins.     

Characterization of the Ehrlich ascites tumor plasma lipoproteins.

1. The lipoproteins of the Ehrlich ascites tumor plasma were separated into 3 distinct fractions, very low density, low density and high density lipoproteins by preparative ultracentrifugation combined with agarose column chromatography. 2. High density lipoproteins contained 74% of the total protein in the lipoproteins. By contrast, most of the lipids were present in the very low density lipoprotein fraction. 3. The fatty acid compositions of the cholesteryl esters were appreciably different in the very low, low and high density lipoproteins, whereas phospholipid and triacylglycerol fatty acid compositions were quite similar in the 3 lipoprotein fractions. 4. Very low and high density apoprotein electrophoretic patterns on sodium dodecyl sulfate-acrylamide gels were similar to those observed in the corresponding lipoprotein fractions obtained from other mammalian species. The low density fraction, however, contained 7 apoprotein bands, and 32% of the low density apoprotein was soluble in tetramethyl urea. 5. The average molecular weights as determined by analytical ultracentrifugation were 2-10(7) (very low density), 6-10(6) (low density) and 4.4-10(5) (high density).     

Differential scanning calorimetric studies of native and freeze-damaged very low density lipoproteins in hen’s egg yolk plasma

Lipid thermal transition patterns of the very low density lipoproteins in native and variously treated egg yolk plasma and extracted total very low density lipoproteins lipids have been recorded by differential scanning calorimetry in the temperature range 220–300 K, after lowering the freeze endotherm of free water in the sample with ethylene glycol. Three distinguishable patterns of lipid endotherms, designated types 1, 2 and 3 were obtained, respectively, from (i) native very low density lipoproteins in egg yolk plasma, (ii) freeze damaged very low density lipoproteins in gelled egg yolk plasma and (iii) extracted total lipids of very low density lipoproteins dispersed in water. Protein-depleted ‘lipid core’ particles of very low density lipoproteins obtained by exhaustive proteolysis of egg yolk plasma gave type 2 lipid transition pattern suggesting similarities in its lipid association with that of the freeze damaged very low density lipoproteins. Freezing the ‘lipid cores’ of very low density lipoproteins led to phase separation and gave type 3 lipid transition pattern of water-dispersed, phase-separated total very low density lipoprotein lipids. Relative heat uptake of native very low density lipoproteins in egg yolk plasma was about 15% lower than the freeze damaged sample or of the extracted total lipids. Treatments which prevented aggregation and gelation of very low density lipoproteins in egg yolk plasma during frozen storage, namely with additives such as glycerol or NaCl, gave subsequent lipid transition pattern intermediate between type 1 and 2, indicating that while very low density lipoprotein aggregation is prevented, additives do not altogether prevent changes in lipid association in these particles.     

Selective accumulation of low density lipoproteins in damaged arterial wall

To determine whether damaged arterial wall selectively accumulates lipoproteins, normocholesterolemic rabbits were injected with human radiolabeled low density lipoproteins, high density lipoproteins, and/or albumin 24 hr to 12 weeks after balloon-catheter de-endothelialization of the abdominal aorta. When 125I-labeled low density lipoproteins and 99mTc-labeled albumin were injected simultaneously, the amount of 125I-low density lipoprotein present 24 hr later in abdominal aortas increased steadily, for several weeks, above the amount present at 24 hr in control animals. The increase correlated closely with the degree of re-endothelialization and correlated closely with the degree of re-endothelialization and reached an average maximum for the whole abdominal aorta of three times control when re-endothelialization was between 75 and 85% complete. By contrast, the amounts of 99mTc-albumin or 125I-labeled high density lipoprotein in balloon-damaged abdominal aortas, and the amounts of 125I-low density lipoprotein, 125I-high density lipoprotein, or 99mTc-albumin in undamaged thoracic aortas of injured animals showed no such increase. As early as 2 weeks after de-endothelialization, en face radioautographs made following injection of 125I-labeled low density lipoproteins revealed localized areas of greatest radioactivity around the leading edges of regenerating endothelial islands, broad areas of intermediate radioactivity corresponding to the de-endothelialized areas, and very like radioactivity in the re-endothelialized areas. This pattern occurred rarely with 125I-labeled high density lipoproteins and not at all with 125I-labeled albumin. The results suggest that low density lipoproteins are selectively accumulated by the healing rabbit aorta and that the accumulation is greatest in regions where the endothelium is actively regenerating.     

Cerebrosides of human aorta: isolation, identification of the hexose, and fatty acid distribution

Cerebrosides have been isolated from adult human aortic tissue. Each aorta was divided into portions classified as normal, fatty streaks, fibrous plaques, or complicated lesions. The cerebrosides were isolated by Florisil column chromatography, mild alkaline methanolysis, a second Florisil column, and preparative thin-layer chromatography. The concentration of cerebrosides was higher in fatty streaks than in the more advanced plaques; apparently normal tissue gave the same cerebroside content as plaques found in the same aorta. The quantities of cerebrosides ranged from 0.01 to 0.73% of the total lipid. Of the 16 cerebroside samples isolated, 10 contained glucosyl ceramide, 1 contained galactosyl ceramide, and 5 were not analyzed for specific hexose. The fatty acid distribution was determined for 11 of the samples; it was similar to that of spleen cerebrosides. We suggest that aortic cerebrosides originate in the plasma. "Normal tissue" cerebrosides contained less unsaturated fatty acid than cerebrosides from a diseased area of the same aorta. Preparative thin-layer chromatography, the last step of cerebroside isolation, always separated at least two unidentified substances. One of these substances yielded both glucose and galactose on acid hydrolysis. Their removal from the cerebrosides accounts for the lower values for cerebroside compared to other authors' determinations.     

Seasonal variations of plasma lipids and lipoproteins in the hedgehog, an animal model for lipoprotein (a) metabolism: relation to plasma thyroxine and testosterone levels

We describe a study of the seasonal variations of hedgehog plasma lipids and lipoproteins and their correlation with changes in the activities of the thyroid and testis. In ten male hedgehogs, plasma concentrations of lipids, thyroxine and testosterone were assayed each month for 1 year beginning in September, while plasma lipoproteins from five of these animals were analyzed at the same dates using density gradient ultracentrifugation. All classes of plasma lipids (cholesterol, total glycerol and phospholipids) exhibited statistically significant seasonal variations in their respective concentrations, with simultaneous maxima (cholesterol: 207 +/- 39 mg/100 ml; total glycerol: 50 +/- 9 mg/100 ml; phospholipids: 266 +/- 25 mg/100 ml) during late fall-early winter, i.e., during the period of the year when plasma levels of both thyroxine and testosterone were minimal. Plasma lipids subsequently decreased to minimal levels either in early summer (cholesterol: 129 +/- 18 mg/100 ml; phospholipids: 178 +/- 20 mg/100 ml) or in late winter (total glycerol: 22 +/- 9 mg/100 ml). Very low density lipoproteins (d less than 1.015 g/ml) were found at low levels (less than 15 mg/100 ml) during the cold months, and then became detectable as trace components only. The total concentration of the mixed lipoprotein population (i.e., low density lipoproteins, Lp(a), and high density lipoprotein (HDL)-like particles) in the d 1.015-1.065 g/ml interval decreased by almost 50% from January to February (from 164.3 to 89.2 mg/100 ml), i.e., following a 10-fold increase in the level of plasma testosterone, and immediately before the rapid doubling in plasma thyroxine concentration. The staining intensity of the electrophoretic band with migration characteristics corresponding to those of Lp(a) decreased considerably during winter. At the same period of the year, lower density (1.032-1.055 g/ml) HDL-like particles disappeared. The concentration of lipoproteins with d 1.065-1.162 g/ml, which included Lp(a) particles in addition to typical HDL, equally underwent seasonal variations. These variations consisted of two successive maxima in late fall (426.4 mg/100 ml) and late winter (458.3 mg/100 ml) with two subsequent decreases leading to minima in February (327.8 mg/100 ml) and August (257.1 mg/100 ml). Finally, very high density lipoproteins (d 1.162-1.259 g/ml) were heterogeneous, containing both cholesterol-rich (d 1.162-1.227 g/ml) and phospholipid-rich (d 1.194-1.259 g/ml) subpopulations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Utilization of free fatty acids complexed to human plasma lipoproteins by mammalian cell suspensions

The purpose of this study was to determine whether lipoprotein-bound free fatty acid could be utilized by isolated mammalian cells. Ehrlich ascites tumor cells were incubated in vitro with radioactive free fatty acids that were bound to human plasma lipoproteins. Under these conditions, lipoprotein-bound free fatty acids were readily taken up by the cells. After 2 min of incubation with free fatty acids bound to low density lipoproteins, most of the radioactivity that was associated with the cells was in the form of free fatty acids. As the incubation continued, increasing amounts of radioactivity were incorporated into CO(2) and cell lipids, particularly phospholipids. Most of the free fatty acid uptake was the result of fatty acid transfer from low density lipoproteins to the cell, not from irreversible incorporation of the intact free fatty acid-low density lipoprotein complex. Fatty acid uptake increased as the ratio of free fatty acid to low density lipoprotein was raised. When albumin was added to the medium, free fatty acid uptake decreased. A large percentage of the newly incorporated cellular radioactivity was released into the medium if the cells were exposed subsequently to a solution containing albumin. Most of the released radioactivity was in the form of free fatty acid. The results with this experimental model suggest that lipoprotein-bound free fatty acid, like albumin-bound free fatty acid, is readily available for uptake by isolated cells. The mechanism of free fatty acid utilization by the Ehrlich cell is similar when either low density lipoprotein or serum albumin serves as the fatty acid carrier.     

Influence of lysophosphatidylcholine on the metabolism of plasma lipoproteins.

Both low density lipoproteins and cellular membranes are known to have a high affinity for lysophosphatidylcholine. In this study lysophosphatidylcholine influenced the retention of lipoproteins by arterial tissue in vitro and the rate of disappearance of low density lipoproteins from the blood in vivo. Pieces of aorta from rabbits or rhesus monkeys were successively incubated for 90 min each in 2 or 3 solutions. After the last incubation the intima plus inner media was dissected from the remainder of the aorta for analysis. The second incubation always contained lipoproteins labeled with [3H]leucine. When lysophosphaticylcholine was included in the first but not in the second incubation fluid, the retention of low, or high density lipoproteins by the intima plus inner media increased. A subsequent incubation of the piece of artery in a fluid with trypsin or lysophosphatidylcholine caused a release of some of the lipoproteins. Lysophosphatidylcholine was bound simultaneously by plasma low density lipoproteins and vascular tissue in vitro and appeared to promote the association of the latter two components. When lysophosphatidylcholine equal to 2--10 times the usual total intravascular content was injected intravenously into control squirrel monkeys or rabbits, it was rapidly cleared from the blood. On the other hand, injected lysophosphatidylcholine persisted in the blood of hyperlipoproteinemic rabbits and was associated with the low density lipoproteins. In control animals, the injection of lysophosphatidylcholine was associated with an increase in the rate of removal of 125I-labelled low density lipoprotein from plasma and of its appearance in liver.     

Does a HDL injection reduce the development of serum hyperlipidemia and progression of fatty streaks in cholesterol fed rabbits?

The influences of homologous (rabbit) or heterologous (human) high density lipoprotein (HDL) on the development of serum hyperlipidemia and progression of fatty streaks were studied in cholesterol fed rabbits. Three groups of New Zealand rabbits were fed a 0.5% cholesterol rich diet for 8 weeks. Additionally into these animals the following solutions were injected intravenously two times per week: group 1 (control): saline; group 2: human HDL dissolved in saline; group 3: rabbit HDL dissolved in saline. The animals of group 2 had lower serum cholesterol levels during the dietary period than rabbits of group 1 (p < 0.05) but the surface of intima covered with fatty streaks was the same as in group 1. On the other hand, the serum cholesterol level in rabbits of group 3 was the same as in group 1 during the whole experimental period, but the surface of aorta covered with fatty streaks was significantly lower (p < 0.05) in group 3 than in group 1. The results of this study support the hypothesis of an antiatherogenic action of HDL, which seems to be independent of the influence of HDL on the serum lipids but depends on the source of HDL.