In vitro glycation of very low density lipoprotein (VLDL) reduced the susceptibility to lipoprotein lipase (LPL) as the level of glycation increased. Addition of reduced glutathione to an incubation medium of serum and glucose interfered with glycation of serum proteins when the concentration of reduced glutathione was higher than 10 mM. At concentrations higher than 25 mM, it also significantly prevented the glycation induced reduction of fatty acid releases from VLDL by LPL. There were no such effects on glycation of serum protein and the fatty acid release from the addition of aminoguanidine. By contrast, addition of Dlysine enhanced glycation of serum proteins by glucose and further decreased fatty acid release from VLDL by LPL. From these results, it is suggested that glycation of VLDL decreases the susceptibility of VLDL to LPL. Delayed catabolism of VLDL in diabetic patients is considered partly caused by glycation of apoproteins, which renders VLDL less sensitive to LPL, in addition to the decreased LPL activity in diabetes mellitus.
To evaluate the relationship between human body composition and serum lipids levels, the distributions of subcutaneous adipose tissue (AT) and muscle thickness were evaluated in Japanese 449 males and 542 females, aged from 35 to 77 years. Among males, a significant positive correlation was observed between AT thicknesses and total cholesterol, and negative relationships between the AT and HDL-C as well as HDL-C/TC ratio. Among females, similar but weaker relationships were found for AT at the upper arm and trunk sites. However, the thigh AT thickness was positively correlated with HDL-C and HDL-C/TC ratio only among women. The muscle thickness in the abdomen and the thigh correlated significantly with HDL-C/TC for both sexes. Furthermore, the regional trend observed in both sexes remained significant after correction for concomitant variables such as age, tobacco and alcohol intake. We conclude that it is necessary to evaluate not only total body fat but muscle and AT thickness distributions when evaluating the relationship between body composition and serum lipids and lipoproteins.
Apolipoprotein E (apoE) in high density lipoprotein (HDL) fraction (HDL-fr) was determined by the immunofixation method and turbidimetric immunoassay (TIA) after precipitation with phosphotungstic acid/MgCl2 in normolipidemic control subjects and patients with type IV hyperlipemia and hyper HDL-cholesterolemia. Immunofixation assay revealed two major bands of apoE in whole serum : one in the α-area and the other in the preβ-al-ea. ApoE in α-area (α-apoE) was identical to α-apoE in HDL-fr separated by ultracentrifugation but not to α-apoE in HDL-fr separated by precipitation (pHDL-fr). α-ApoE in pHDL-fr lacked the slower area of the band. Agarose column chromatography and gradient gel electrophoresis indicated that α-apoE belongs to early fractionated HDL, and that the precipitatable α-ApoE belongs to the higher molecular size HDL. α-ApoE (%) estimated by immunofixation showed a strong positive correlation with apoE (%) in pHDL-fr. ApoE (%) in pHDL-fr was higher in case of hyper HDL-cholesterolemia and lower in type IV hyperlipemia than in controls, and was inversely correlated with serum triglycerides (TG) and positively with HDL-cholesterol (especially HDL2-cholesterol) in these subjects. It is suggested that the variation of apoE in pHDL-fr depends on the level of HDL2. Also, it may be suggested that apoE in pHDL-fr and precipitatable α-apoE belong to low and high molecular HDL2, respectively, and that apoE content in these particles is correlated with HDL2.
Type III is a remnant hyperlipoproteinemia identified by the presence of β-VLDL (remnant lipoprotein) as well as a genetic variant of apo E (apo E2/2). The RLP isolated from the serum of Type III patients by a new method we have developed, the RLPcholesterol assay, was identified as chylomicron and VLDL remnant. In addition, the RLP-C levels of the Type III patients were significantly higher than other hyperlipidemic patients with similar serum TG levels, while the ratio of TC/TG in RLP-C of both groups was not significantly different. The RLP-cholesterol assay appears to be useful for the screening and monitoring of Type III hyperlipoproteinemia when used in conjunction with the assays of serum TG level and genetic apo E isoform analysis.
Lipoprotein (a) (Lp (a)) is a plasma lipoprotein of high atherogenicity and competes with plasminogen at the site of plasminogen receptors. It is known that diabetic patients show a hypercoagulable state which might contribute to diabetic vascular complications. In the present study, mean levels of plasma Lp (a) and parameters of coagulation and fibrinolysis such as thrombin-antithrombin III complex (TAT) and α2 plasmin inhibitor-plasmin complex (α2PIC) were elevated in diabetic patients with nephropathy compared to healthy controls. A significant positive correlation was observed between the plasma levels of Lp (a) and α 2PIC (p<0.05). Plasma levels of a 2PIC showed a significant positive correlation with those of TAT in the diabetic group, while there was no significant correlation observed in the non-diabetic group. The present results suggest that factors of Lp (a) and coagulation-fibrinolytic systems interacted, contributing to vascular complications in diabetic patients with nephropathy.
The effect of protease inhibitors, leupeptin and pepstatin A, on the metabolism of acetylated low density lipoprotein (acetyl-LDL) was investigated in cultured rat peritoneal macrophages and compared with that of chloroquine. While both leupeptin and pepstatin inhibited the proteolytic degradation of 125I-acetyl-LDL, a combination of both showed an additive effect. Similar to chloroquine, both protease inhibitors diminished [3H] oleate incorporation into cellular cholesteryl [3H] oleate and increased cholesterol content of macrophages. These results suggest that both thiol protease and cathepsin D participate in the physiological degradation of apolipoprotein in macrophages. The inhibition of apolipoprotein degradation seemed to have an effect on cholesterol metabolism in macrophages cultured with acetyl-LDL.
To investigate changes in the major components of atherosclerotic lesions during the progression of this disease, we measured the lesional areas of macrophages, smooth muscle cells, collagen fibers, and extracellular lipid deposits in the aortas of WHHL rabbits. Aortic segments with lesions of various stages were stained for histological and immunohisto-chemical examination, and the area of each lesional component was measured by a color image analyzer. In the early fatty streaks observed in 3-month-old rabbits, macrophages were predominant in the intima and were also observed in the inner layer of the media. In the transitional lesions (fibro-fatty streaks) found in rabbits at 11 to 15 months of age, an increase in the lesional area of macrophages was prominent compared to other lesional components. Thus, macrophages may play an important role in the progression of aortic atherosclerosis at this stage. In advanced complicated lesions observed in rabbits at 20 to 24 months of age, the area of macrophages and smooth muscle cells did not increase, whereas the area of collagen fibers and extracellular lipid deposits increased. Therefore, both the disruption of foam cells and fibrosis may play an important role in the progression of atherosclerosis at this stage.
Cell degeneration and collagenosis are the main features in atherosclerotic plaque. We examined the effects of human low density lipoprotein (LDL) on cultured rabbit aortic smooth muscle cells (SMCs). Copper oxidized LDL (LDL+Cu) injured the SMCs more than did native LDL. Cytotoxicity of oxidized LDL was prevented by the simultaneous addition of butylated hydroxytoluene (BHT) and ethylenediamine tetraacetic acid. Collagen synthesis increased up to 6 fold after incubation with 200μg protein/ml of both native and oxidized LDL compared with that incubated in bovine serum albumin. Noncollagen protein synthesis was significantly reduced by oxidized LDL when compared to that by native LDL. Therefore, oxidized LDL increased the relative collagen synthesis (3.33%) to a greater extent than did native LDL (0.72%). By adding BHT to LDL+ Cu, the elevated relative collagen synthesis was reversed due to the restoration of noncollagen protein synthesis while it also inhibited LDL peroxidation as evaluated by the formation of malondialdehyde (MDA). However, sodium MDA (up to 200 μM) did not induce either cytotoxicity or an increase of relative collagen synthesis. We therefore conclude that oxidized human LDL enhanced the relative collagen synthesis coinciding with the induction of injury in cultured aortic SMCs, however free MDA may not be the component responsible for these effects.
During re-endothelialization after intimal denudation induced by balloon catheter in the rat aorta, re-formation of the basement membrane (BM) was examined in samples taken 15 min, 3, 7, 14, 21 and 28 days after the balloon-induced injury. Although the luminal surface of the aortic wall was covered by round-shaped regenerating endothelial cells (ECs) by 7 days after intimal denudation, no continuous BM structure was detectable until 21 days. Periodic acid- thiosemicarbazide gelatin-methenamine silver (PATSC-GMS) staining for electron microscopy and immunostaining of type IV collagen and laminin demonstrated the accumulation of BM components underneath the regenerating ECs after 14 days. The expression of type IV collagen mRNA was revealed in regenerating ECs by in situ hybridization. A continuous BM structure first appeared 21 days after intimal denudation and was almost complete by 28 days. Simultaneously, the regenerating ECs flattened and attached more closely to the BM than in earlier phases. In conclusion, we consider that the regenerating ECs produce the BM components and suggest that reorganization of the newly formed BM is important in the process of differentiation of regenerating ECs.