Low-density Lipoprotein Activity Research Articles

Effects of phospholipase A2 and its products on structural stability of human LDL: relevance to formation of LDL-derived lipid droplets

Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. To determine the effects of FFA on these transitions, we hydrolyzed LDL by phospholipase A(2) (PLA(2)), removed FFA by albumin, and analyzed structural stability of the modified lipoproteins. Earlier, we showed that heating induces LDL remodeling, rupture, and coalescence into lipid droplets resembling those found in atherosclerotic lesions. Here, we report how FFA affect these transitions. Circular dichroism showed that mild LDL lipolysis induces partial β-sheet unfolding in apolipoprotein B. Electron microscopy, turbidity, and differential scanning calorimetry showed that mild lipolysis promotes LDL coalescence into lipid droplets. FFA removal by albumin restores LDL stability but not the protein conformation. Consequently, FFA enhance LDL coalescence into lipid droplets. Similar effects of FFA were observed in minimally oxidized LDL, in LDL enriched with exogenous FFA, and in HDL and VLDL. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA.

Beneficial effects of fish liver preparations of sea bass (Lates calcarifer) versus gemfibrozil in high fat diet-induced lipid-intolerant rats.

Abnormal lipid metabolism is a main cause of dyslipidemia, which is a major risk factor for coronary heart disease and obesity and is even linked to diabetic-dyslipidemic complications. Fifteen days of high-fat feeding in Charles Foster rats resulted in a significant increase in baseline serum lipid levels accompanied by pronounced dyslipidemia. Treatment with fish liver preparations (FLPs) from sea bass and the standard drug gemfibrozil produced a lowering of serum lipids and glucose levels, along with a fall in very-low-density and low-density lipoprotein and an increase in high-density lipoprotein levels. Simultaneously, reactivation of plasma postheparin lipolytic activity (PHLA) and lecithin:cholesterol acyltransferase (LCAT) activity was also observed. A positive correlation was observed between low-density lipoprotein activity and fecal bile acid excretion, which was enhanced on treatment with FLPs and gemfibrozil, indicating the catabolic process for normal lipids and cholesterol homeostasis. These data suggest that FLPs and gemfibrozil not only lower lipid intolerance but also reduce diabetic-dyslipidemic complications by activating peroxisome proliferator-activated receptors (PPAR).

Soybean Protein Products as Regulators of Liver Low-Density Lipoprotein Receptors. II. α−α‘ Rich Commercial Soy Concentrate and α‘ Deficient Mutant Differently Affect Low-Density Lipoprotein Receptor Activation

Activation of low-density lipoprotein (LDL) receptors has been decribed in a human hepatoma cell line (Hep G2) exposed to the 7S globulin from soy (Lovati et al. J. Agric. Food Chem. 1998, 46, 2474−2480). This in vitro model supports a direct activity of soy protein component(s) in lowering LDL cholesterolemia. To ascertain whether α or α‘ subunits from 7S globulin, or both, are responsible for the biochemical effect, a mutant soy cultivar, devoid of the α‘ subunit (Keburi), was evaluated in the same model. A commercial isoflavone-poor, heat-hydrolyzed soy preparation found effective in human hypercholesterolemia (Croksoy) was also tested. While Croksoy proved to have a similar activity as the whole 7S globulin, the α‘-free Keburi variant showed no effect on LDL receptor activation. These findings support the reliability of the human liver cell model in predicting a human hypocholesterolemic activity and suggest that specific peptide(s) may be responsible. Furthermore, they indicate that some soy cultivar...

Total antioxidant activity of low density lipoproteins and the relationship with α-tocopherol status

A rapid method is described for measuring the antioxidant activity of low density lipoproteins. Studies were undertaken on individuals attending a hyperlipidaemia clinic, an unsupplemented group and a group after supplementation with 300 mg dl-α-tocopherol acetate for nine weeks. The results show a positive correlation between the antioxidant activity and α-tocopherol content of LDL in the supplemented group.

Cutaneous Xanthomas in Homozygous Familial Hypercholesterolemia

Figure 1. A baby boy was found to have familial hypercholesterolemia at the age of six months. His parents also had hypercholesterolemia. Seven and a half years later, studies of the activity of low-density lipoprotein (LDL) receptors in proliferating lymphocytes indicated that the parents were heterozygous and the child was homozygous for LDL-receptor deficiency. The child's serum cholesterol concentration ranged from 850 to 1000 mg per deciliter (22 to 26 mmol per liter), and he had cutaneous xanthomas on his knees (as shown), elbows, and interdigital folds. Because the hypercholesterolemia was refractory to treatment with lovastatin and cholestyramine, the patient . . .

Platelets in patients with homozygous familial hypercholesterolemia are sensitive to Ca(2+)-mobilizing activity of low density lipoproteins.

While some data suggest that Ca(2+)-mobilizing effects of low density lipoprotein (LDL) in platelets are mediated by a specific membrane receptor, the data about the nature of this lipoprotein-binding site are contradictory. This work was performed in order to assess possible involvement of apolipoprotein (apo) B,E receptor, present in most cell types. To answer the question we compared effects of LDL in normal platelets and those obtained from patients with homozygous familial hypercholesterolemia (HFH), characterized by absence of functional apo B,E receptors. We have found that in accordance with previous results LDL induced instant reversible elevation of free cytoplasmic calcium concentration ([Ca2+]i) in fura-2-loaded platelets. The effect was observed both in healthy and HFH groups. Neither half-maximal effective concentrations nor maximal effects of LDL differed significantly between two groups. Ca(2+)-mobilizing effects of lipoproteins were potentiated about 4-fold by epinephrine and completely blocked by prostaglandin E1 both in platelets of healthy and HFH subjects. The similarity of lipoprotein effects in control and HFH platelets is evidence that apo B,E receptor does not mediate the Ca(2+)-mobilizing activity of LDL in this cell type.

Platelet-activating factor acetylhydrolase in plasma lipoproteins from patients with ischemic stroke.

Platelet-activating factor is a potent bioactive phospholipid and may play an important role in occlusive vascular diseases. To assess the inactivation of this autacoid in plasma, we measured platelet-activating factor acetylhydrolase activity in plasma low density and high density lipoproteins from patients with ischemic stroke. Low density and high density lipoproteins were separated by ultracentrifugation from plasma of 33 patients with cerebral thrombosis and 31 age-matched healthy control subjects, and platelet-activating factor acetylhydrolase activity in each fraction was assayed. The average values of platelet-activating factor acetylhydrolase activity in low density lipoprotein from patients and control subjects were 41 +/- 18 and 29 +/- 17 nmol/ml per minute, respectively, and the difference was statistically significant (p less than 0.01 U test). There was no difference in activity in high density lipoprotein between the two groups (16 +/- 11 versus 14 +/- 9 nmol/ml per minute, respectively). The increased plasma platelet-activating factor acetylhydrolase activity in stroke patients is primarily attributable to the increased binding to low density lipoprotein, and this increase may be an adaptation to the augmented generation of platelet-activating factor in ischemic stroke.

Phospholipase A2 activity of low density lipoprotein: evidence for an intrinsic phospholipase A2 activity of apoprotein B-100.

During oxidative modification of low density lipoprotein (LDL) there is extensive degradation of phosphatidylcholine (PtdCho) to lysophosphatidylcholine (lyso-PtdCho), with the removal of fatty acids from the 2 position. The phospholipase A2 (PLA2) activity responsible for hydrolysis is closely associated with LDL. By use of lipoxygenase-oxidized 2-[1-14C]linoleoyl PtdCho as the substrate and delipidated apoprotein B (apo-B), evidence is presented to show that (i) the activity is destroyed progressively during the oxidative modification of LDL; (ii) p-bromophenacyl bromide (pBPB), a histidine modifier that inhibits the oxidative modification of LDL, also substantially inhibits the PLA2 activity; and (iii) photooxidation of LDL in the presence of Rose Bengal completely inactivates the enzyme with concomitant loss of apo-B histidine residues. High molecular weight proteins from delipidated LDL, separated by polyacrylamide gel electrophoresis, showed PLA2 activity. It is suggested that apo-B itself may possess PLA2 activity.

Hemodynamics alter arterial low-density lipoprotein metabolism.

We have investigated the role of hemodynamic factors on low-density lipoprotein transport and metabolism in the intact arterial wall. Freshly excised canine carotid blood vessels were exposed to well-defined pulsatile flow in vitro for continuous periods up to 20 hours. We chose to impose the following hemodynamic conditions on our test carotid arteries: normotension, hypertension (at physiologic flow conditions), and hypertension coupled with elevated flow of canine serum perfusate. In several experiments the effect of endothelial denudation was examined in carotid arteries exposed to normotensive pulsatile flow. A trapped ligand method was used for quantitating low-density lipoprotein uptake and metabolism in the arterial wall. The distribution of both intact and degraded low-density lipoprotein fractions was determined from measurements of radiolabelled low-density lipoprotein activity within thin radial sections of perfused arteries. Our results suggest that both hypertensive hemodynamic simulations exacerbate the uptake of low-density lipoprotein within the arterial wall (by a factor of three to nine). The percentage of low-density lipoprotein that undergoes irreversible degradation falls from 41% under normotensive conditions to below 30% when hypertensive conditions are imposed, indicating that degradative processes are not proportionally elevated with the accelerated influx. A similar pattern is observed for deendothelialized vessels.

Tissue-selective acute effects of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase on cholesterol biosynthesis in lens.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme that regulates cholesterol synthesis, lower serum cholesterol by increasing the activity of low density lipoprotein (LDL) receptors in the liver. In rat liver slices, the dose-response curves for inhibition of [14C]acetate incorporation into cholesterol were similar for the active acid forms of lovastatin, simvastatin, and pravastatin. The calculated IC50 values were approximately 20-50 nM for all three drugs. Interest in possible extrahepatic effects of reductase inhibitors is based on recent findings that some inhibitors of HMG-CoA reductase, lovastatin and simvastatin, can cause cataracts in dogs at high doses. To evaluate the effects of these drugs on cholesterol synthesis in the lens, we developed a facile, reproducible ex vivo assay using lenses from weanling rats explanted to tissue culture medium. [14C]Acetate incorporation into cholesterol was proportional to time and to the number of lenses in the incubation and was completely eliminated by high concentrations of inhibitors of HMG-CoA reductase. At the same time, incorporation into free fatty acids was not inhibited. In marked contrast to the liver, the dose-response curve for pravastatin in lens was shifted two orders of magnitude to the right of the curves for lovastatin acid and simvastatin acid. The calculated IC50 values were 4.5 +/- 0.7 nM, 5.2 +/- 1.5 nM, and 469 +/- 42 nM for lovastatin acid, simvastatin acid, and pravastatin, respectively. Thus, while equally active in the liver, pravastatin was 100-fold less inhibitory in the lens compared to lovastatin and simvastatin. Similar selectivity was observed with rabbit lens. Following oral dosing, ex vivo inhibition of [14C]acetate incorporation into cholesterol in rat liver was similar for lovastatin and pravastatin, but cholesterol synthesis in lens was inhibited by lovastatin by as much as 70%. This inhibition was dose-dependent and no inhibition in lens was observed with pravastatin even at very high doses. This tissue-selective inhibition of sterol synthesis by pravastatin was likely due to the inability of pravastatin to enter the intact lens since pravastatin and lovastatin acid were equally effective inhibitors of HMG-CoA reductase enzyme activity in whole lens homogenates. We conclude that pravastatin is tissue-selective with respect to lens and liver in its ability to inhibit cholesterol synthesis.

Dihomo-γ-Linolenic Acid in Patients with Atherosclerosis: Effects on Platelet Aggregation, Plasma Lipids and Low-Density Lipoprotein-Induced Inhibition of Prostacyclin Generation-Reply

Dihomo-gamma-linolenic acid ( DHLA ), a precursor of monoenoic anti-aggregatory prostaglandins (PGE1, PGD2), was administered for 4 weeks in a daily dose of 1.0 g into 33 patients with atherosclerosis on a basis of a double-blind trial. Comparison of treatment and placebo groups revealed elevation of DHLA in red cell lipids in DHLA -treated subjects. No differences, however, between the two groups could be observed in platelet aggregability, thromboxane A2 generation by platelets, serum cholesterol, PGE1 and PGE2 levels, and in inhibitory activity of low-density lipoproteins against prostacyclin synthetizing system in arteries. The dietary supplementation used did not lead to distinct antithrombotic effects.

培養皮膚線維芽細胞および単球由来マクロファージにおけるコレステロールエステラーゼ活性について

The purpose of the present study was to characterize the properties of cholesterol esterase (CEase) in human skin fibroblasts and monocyte-derived macrophages. Acid and neutral CEases exist in lysosome and microsome, respectively in both cells. Acid CEase activity was higher than neutral CEase activity. Other properties of acid and neutral CEase of fibroblasts and macrophages were similar with respect to Km, effect of phospholipids and divalent cations for phosphatidyl choline (PC) emulsified cholesterol ester (CE).But optimal pH of CE hydrolysing activity in low density lipoprotein (LDL) was located at pH 4.5 and was not detected at neutral or alkaline range, whereas there was two pH optima for CEase activity when PC emulsified CE was used as substrate. This result suggests that LDL-CE hydrolysis can be performed only in lysosome. The uptake and hydrolysis by fibroblasts of 125I-LDL were determined. The uptake of LDL in fibroblasts of familial hypercholesterolemic patients (FHC) was lesser than that of normals, but the hydrolysis of LDL was increased. The amount of uptake of a-LDL in macrophages of FHC was not different from that of normals, but the hydrolysis of LDL was decreased.

In Vivo Suppression of the Primary Immune Response by a Species of Low Density Serum Lipoprotein

An apparent subspecies of normal human serum low density lipoprotein (LDL-In) has been identified with suppressive activity for early or facilitating events of human lymphocyte mitogen and allogenic cells stimulation in vitro. This report describes the effects of in vivo administration of LDL-In on the mouse anti-SRBC immune response. Human LDL-In is not species specific and was capable of suppressing the in vivo mouse anti-sheep erythrocyte (SRBC) hemagglutination response by 88% after the administration of 500 to 600 mug LDL-In IV, whereas human serum high density lipoproteins and fibrinogen had no effect. Maximal suppression occurred only when LDL-In was injected 24 to 48 hr before antigen administration. Simultaneous or subsequent injection of LDL-In had no effect. The activity of LDL-In was influenced by antigen dose and maximal at low antigen doses. The number of splenic plaque-forming cells was also reduced indicating a suppression of the clonal expansion of primary B cells to antibody-secreting cells rather than only suppression of antibody synthesis by differentiated B cells and their progeny. These observations suggest the hypothesis that endogenous LDL-In could play an important immunoregulatory role in the maintenance of immune homeostasis and the "natural" suppression of non-productive lymphocyte proliferation.