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PubMed1996

[LDL oxidation in homozygous familial hypercholesterolemia: effects of selective LDL-apheresis treatment].

Homozygous familial hypercholesterolemia (HFH) results from a mutation affecting both the structure and function of a cell surface receptor that removes low density lipoproteins (LDL) from plasma. The disorder is characterized by autosomal dominant inheritance, a lifelong elevation in the concentration of LDL-bound cholesterol in blood and by cholesterol deposits that form xanthomas and early coronary artery disease. HFH patients, as a result of the increased levels and prolonged residence time of LDL in plasma, have a strong tendency toward accumulation of LDL-cholesterol in the arterial wall causing premature atherosclerosis. Selective LDL-apheresis (LA) on dextran/sulphate cellulose columns is the best therapy reducing mortality of these patients. We previously showed that prolonged lifelong enhanced LDL oxidation in HFH. LDL undergo oxidation before being taken up by macrophages then transformed into foam cells. At the present time, the relevance of the in vitro macrophages studies to the accumulation of cholesterol esters in scavenger cells of HFH patients is not yet established. The aim of this study was to investigate LDL oxidation, induced by xanthine (2 mM)+xanthine oxidase (100 mU), and cholesterol esterification in macrophages, in 8 HFH patients before and after LA. LDL peroxidation by conjugated-diene absorbance showed an increased resistance against oxidation after LA: lag time 129 +/- 25 vs 112 +/- 27 min, p < 0.05; diene production 9.1 +/- 2.1 vs 13.9 +/- 2.5 nM/min/mg LDL, p < 0.01. Peroxidation was also evaluated from lipid peroxides (158 +/- 34 vs 57 +/- 18 nM/mg protein after LA, p < 0.05) and malonyldialdehyde (38 +/- 12 vs 27 +/- 8 nM/mg protein after LA, p < 0.05) content. When oxidized LDL was run on polyacrylamide gel extensive apo-B100 fragmentation was observed in LDL before LA, vs a less fragmentation after LA. A similar reduction was obtained in LDL agarose mobility after LA (1.7 +/- 0.2 vs 2.5 +/- 0.2, p < 0.05). Cholesterol esterification in mouse peritoneal macrophages was also decreased after LA (8.5 +/- 1.8 vs 14.6 +/- 2.7 nM/mg cell protein/12 hours, p < 0.05). Vitamin E content of LDL (mg/g protein) was increased after LA (4.44 +/- 1.0 vs 3.9 +/- 1.2, p < 0.05). Thus, selective LA, not only decreases the pool of LDL, but it also induces changes that render LDL less susceptible to oxidation and decreased high cholesterol esterification in macrophages. The prevention of these mechanisms by LA contributes actively to retard atherogenesis in HFH patients.

Analysis of Variance↗

PubMed1996

Effect of lipid transfer activity and lipolysis on low density lipoprotein (LDL) oxidizability: evidence for lipolysis-generated non-esterified fatty acids as inhibitors of LDL oxidation.

Low density lipoproteins (LDL) were modified in vitro in the presence of lipid transfer activity and lipolysis, which induced alterations in the size and lipid composition of LDL particles but not in their antioxidant content. Subsequently, modified LDL were oxidized with copper sulfate and the extent of LDL oxidation was evaluated. Lipid transfer activity alone, or in combination with lipolysis, led to a significant reduction of LDL oxidability as compared with starting homologous LDL. Furthermore, the combined effect of lipid transfers and lipolysis reduced LDL oxidability to a significantly greater extent than did lipid transfers alone. Consistent results were obtained by measuring either the formation of lipid peroxides, the appearance of thiobarbituric acid reactive substances (TBARS), the disappearance of polyunsaturated fatty acids (PUFA), or the generation of cholesterol oxides. Non-esterified fatty acids (NEFA) arose as putative candidates in reducing oxidation susceptibility of LDL: NEFA-containing LDL were less oxidizable; the enrichment of LDL with either oleic acid or linoleic acid reduced significantly their oxidability; the oxidation susceptibility of either in vitro modified LDL or LDL isolated from normal or analbuminemic patients significantly increased after reduction of their NEFA content with fatty acid-poor albumin. After NEFA depletion, small-sized LDL resulting from the combined effects of lipid transfer and triglyceride hydrolysis activities became more oxidizable than large-sized LDL treated with lipid transfer activity alone. In addition, the PUFA to total fatty acid ratio and the oxidability of modified LDL varied accordingly after NEFA depletion, showing that in the present study not only lipoprotein-bound NEFA but also the total fatty acid composition of LDL could account for alterations in their oxidability.

Chromatography, High Pressure Liquid↗

PubMed2005

Beneficial effects of atorvastatin on sd LDL and LDL phenotype B in statin-naive patients and patients previously treated with simvastatin or pravastatin.

BACKGROUND: The presence of increased levels of small dense (sd) LDL (phenotype B) is associated with a substantial increase of cardiovascular disease risk. Since lowering of plasma low-density lipoprotein-cholesterol (LDL-C) by statins involves an up-regulation of the LDL receptor, we questioned whether LDL lowering by atorvastatin affects different LDL subfractions equally. METHODS: Fifty-four hypercholesterolemic patients, requiring treatment for prevention of coronary heart disease received atorvastatin (10, 20 or 40 mg/day), either as initial therapy (n=33), or as replacement therapy (n=21) for pravastatin or simvastatin (both at 40 mg/day). In addition to plasma lipid measurements, cholesterol LDL subfractions were separated and analysed before and after 3 months of treatment. RESULTS: In addition to the expected LDL-C decrease (-34%; p<0.0001), a major reduction in sd LDL occurred after atorvastatin therapy (-38.2%; p<0.0001). Interestingly, sd LDL decreased as much in patients previously treated with other statins (-36%; p<0.002). A close correlation (r=0.89, p<0.001) was found between reduction of sd LDL and that of LDL-C, in patients with phenotype B. Although high-density lipoprotein-cholesterol (HDL-C) was not affected by atorvastatin treatment, plasma triglycerides decreased by 27.4% (p<0.0001). Only a weak correlation (r=0.35, p<0.01) was found between the reduction of plasma triglycerides and the decrease of sd LDL after atorvastatin treatment. CONCLUSION: These results show that the reduction of LDL-C by atorvastatin largely reflects a lowering of sd LDL. Our data also suggest that triglyceride lowering plays only a partial role in sd LDL reduction.

Adolescent↗

PubMed2001

Assessment of the fifth ligand-binding repeat (LR5) of the LDL receptor as an analytical reagent for LDL binding.

The fifth ligand binding repeat (LR5) of the low density lipoprotein (LDL) receptor was assessed ex vivo as an 'analytical reagent' to distinguish LDL state, in atherosclerosis risk monitoring. LR5 was immobilized to mercaptoundecanoic acid modified gold surfaces via a glycine linker. Surface plasmon resonance (SPR) was used to monitor LDL binding. Unfolded LR5 was ineffectual as an affinity ligand for LDL but refolded LR5 showed a high affinity for native LDL but little affinity for oxidized LDL. LR5 refolded in the presence of calcium or EDTA gave the equivalent LDL binding capacity. However, EDTA-LR5 was less stable than Ca-LR5 at pH 5 and, from tryptophan fluorescence evidence, they appeared to involve different regions of LR5 and/or LDL in the binding. Involvement of amino acid residues of the calcium cage of LR5 was tested in LDL binding by monitoring calcium ion release with a calcium ionophore. The results were consistent with approximately 7-8 LR5 binding per LDL, of which only some induce calcium release (a maximum of approximately 25 mol% calcium, based on LR5, was released during LDL binding). For LDL binding to the LDL receptor in vivo more than one ligand-binding repeat is needed and this may be consistent with LR5 acting here also at binding sites which other LRs normally occupy in the LDL-LDL receptor complex. This initial study is encouraging for the use of a minimum peptide repeat array based on the conserved region of the LRs as an affinity surface for atherosclerosis risk monitoring.

Amino Acid Sequence↗

PubMed1998

LDL hypercholesterolemia is associated with accumulation of oxidized LDL, atherosclerotic plaque growth, and compensatory vessel enlargement in coronary arteries of miniature pigs.

The association between accumulation of oxidized low density lipoprotein (LDL) and (1) progression of atherosclerotic plaques and (2) compensatory enlargement was assessed in the coronary arteries of LDL-hypercholesterolemic miniature pigs. In miniature pigs fed a 4% cholesterol diet, LDL cholesterol levels increased from 27+/-3.5 mg/dL (mean+/-SEM, n=36) to 250+/-28 mg/dL (n=10), 260+/-15 mg/dL (n=6), and 260+/-17 mg/dL (n=10) at 6, 14, and 24 weeks, respectively. Mean intimal areas of lesions in the left anterior descending coronary artery of hypercholesterolemic pigs were 0.16+/-0.046 mm2 at 6 weeks (n=10) and increased 5.4-fold (n=6, P<.05) and 10.6-fold (n=10, P<.001) at 14 and 24 weeks, respectively. Plaque growth was associated with an increase in mean internal elastic lamina area, from 1.44+/-0.17 to 4.38+/-0.52 mm2 (P=.007) and in mean luminal area from 1.42+/-0.15 mm2 in control pigs to 4.38+/-0.52 mm2 in pigs fed a cholesterol diet for 24 weeks (P=.007 vs control). Levels of total LDL in the intima, measured immunocytochemically, were 0.031+/-0.0098, 0.11+/-0.057 (P< or =.05), and 0.43+/-0.082 U (P<.001) at 6, 14, and 24 weeks, respectively. Corresponding levels of oxidized LDL were 0.034+/-0.023, 0.11+/-0.050 (P<.05), and 0.44+/-0.065 U (P<.001), respectively, suggesting that virtually all LDL in the intima is oxidized. Levels of oxidized LDL in the lesions were correlated with the intimal areas (r=.85, P<.0001) but were independent of plasma levels of LDL cholesterol and of oxidized LDL. Plaque levels of oxidized LDL were also correlated with internal elastic lamina areas (r=.72, P<.0001) and with luminal areas (r=.50, P=.0098). Plaque growth in the coronary arteries of LDL-hypercholesterolemic miniature pigs is associated with (1) an increase in plaque levels of oxidized LDL at constant plasma levels of LDL cholesterol and of oxidized LDL and (2) compensatory vessel enlargement proportional to plaque levels of oxidized LDL.

Adaptation, Physiological↗

PubMed2004

RPE cells internalize low-density lipoprotein (LDL) and oxidized LDL (oxLDL) in large quantities in vitro and in vivo.

PURPOSE: To determine whether plasma low-density lipoprotein (LDL) could be internalized by the RPE and which receptors may be involved. A secondary objective was to determine whether ARPE19 cells could be used as a model to investigate cholesterol processing in the RPE. METHODS: Commercially available human LDL was labeled with rhodamine or AlexaFluor 568. Immunofluorescence was performed using commercially available antibodies to LDL-R, CD36, and LOX-1. Cells and tissues were imaged using epifluorescence and confocal fluorescence microscopy. Immunoblot analysis and RT-PCR were performed using published techniques. RESULTS: Intravenously injected rhodamine-labeled LDL (rhoLDL) was detected in the rat RPE by fluorescence confocal microscopy 24 hours after injection. The rhoLDL was present in some areas and absent in others. Cultured ARPE19 cells were also found to internalize LDL and oxidized LDL (oxLDL) readily. Using AlexaFluor 568-labeled LDL we determined that the average cultured RPE cell could internalize approximately 12 to 16 pg of LDL and oxLDL in 24 hours. Immunoblots readily detected the presence of CD36 and LDL-R in the cultured RPE cells but not LOX-1, whereas RT-PCR detected mRNA for all three receptors. Dual-labeling experiments using AlexaFluor 568-labeled LDL and AlexaFluor 488 for the immunolocalization of the receptors showed colocalization of LDL-R with the internalized LDL and CD36 with oxLDL particles. CONCLUSIONS: Plasma LDL readily enters the RPE through the choriocapillaris but is not found homogeneously throughout the retina. This may suggest some form of regulation to the permeability of the fenestrated choroidal endothelial cell junctions. ARPE19 cells are a good model for studying the internalization mechanisms of LDL and oxLDL in vitro. LDL may be used as a vector to carry hydrophobic molecules into the RPE.

Animals↗

PubMed1994

Composition and immunoreactivity of serum low density lipoproteins (LDL) before and after LDL-apheresis on dextran sulfate-cellulose columns.

The changes in low density lipoprotein (LDL) composition and immunoreactivity occurring after LDL-apheresis on dextran sulfate-cellulose columns (DSC) were investigated in 4 hypercholesterolemic patients. After apheretic treatment, serum levels of total cholesterol, triglycerides and apolipoprotein B (apo B) were decreased by 63, 80 and 65%, respectively, whereas the high density lipoprotein (HDL)-cholesterol remained unchanged. At the end of apheresis, LDL contained less triglycerides, more phospholipids and apo E and the ratio of LDL core lipid components, cholesteryl esters and triglycerides, to LDL surface lipid components, unesterified cholesterol and phospholipids was significantly lower. The post-apheretic LDL were characterized by the presence of subfractions slightly larger than those observed in the pre-apheretic LDL. The modifications of the composition and size of LDL after apheresis were accompanied by a relative increase in the immunoreactivity of 4G3 epitope, an apo B epitope located near the LDL-receptor binding site, with no change in the affinity of 1D1, an apo B epitope located in the amino-terminal region of the molecule. The changes in LDL composition, size and immunoreactivity following apheresis, suggest that postapheresis LDL could contain newly synthesized LDL, different from mature LDL. Thus, LDL-apheresis treatment could provide the opportunity to study the structural change of LDL during intravascular metabolism.

Antibodies, Monoclonal↗

PubMed2001

Stimulation of smooth muscle cell proliferation by ox-LDL- and acetyl LDL-induced macrophage-derived foam cells.

To test the hypothesis that LDL lacking of initial oxidation may also anticipate an essential role in the progression for atherosclerotic lesions, we studied the in vitro effect of foam cells induced by low density lipoprotein (LDL), oxidized (ox)-LDL or acetyl-LDL on smooth muscle cell (SMC) proliferation. Intraperitoneal macrophages collected from ICR mice were incubated with buffered saline LDL, ox-LDL or acetyl-LDL to induce foam cell formation. Porcine aortas with atherosclerotic lesions were collected from 5 pigs fed high cholesterol diets. The results indicate that foam cells induced by ox-LDL and acetyl-LDL, but not by LDL, promoted SMC proliferation. SMC proliferation was also increased by ruptured, ox-LDL- and acetyl-LDL- induced foam cells. Immunohistochemically, epitopes of the LDL, ox-LDL, and malondialdelyde (MDA)-LDL were present in atherosclerotic lesions, but the acetyl epitope was not. We suggest that foam cells, whether induced by the oxidized or acetyl or acetyl (unoxidized) form, play an essential role in the pathogenesis of atherosclerosis by stimulating SMC proliferation.

Animals↗

PubMed1992

Monoclonal antibodies against LDL further enhance macrophage uptake of LDL aggregates.

Self-aggregates of low density lipoprotein (LDL) are taken up and degraded more rapidly by macrophages than is native LDL. That enhanced uptake is attributable in part to phagocytosis via the LDL receptor pathway. However, arterial macrophages appear to express little LDL receptor activity. The present studies demonstrate an alternative mechanism by which LDL aggregates could contribute to foam cell formation. This could occur by the formation of large immune complexes that are taken up by macrophages via the Fc receptor. When immune complexes were formed with native, soluble LDL and MB47, a monoclonal antibody specific to the apoprotein B domain recognized by the LDL receptor, the subsequent uptake and degradation of the LDL by macrophages were inhibited 50-80% compared with native LDL alone. In contrast, when aggregated LDL was bound to MB47 at a similar molar ratio, the subsequent degradation of the insoluble immune complexes was two- to fivefold greater than that of aggregated LDL alone. The enhanced uptake was abolished when Fab or F(ab')2 fragments of MB47 were substituted for the intact antibody, indicating that the increased uptake was via the Fc receptor pathway. Furthermore, the uptake of the immune complexes of aggregated LDL was reduced by competition for the Fc receptor with heat-aggregated immunoglobulin. There was also an increase in the rate of cellular cholesterol esterification and an increase in macrophage cholesteryl ester mass. Since aggregates of LDL as well as autoantibodies against modified LDL have been demonstrated in atherosclerotic lesions, it is possible that immune complexes of aggregates of modified LDL may be generated in the intima.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

Effect of fluvastatin slow-release on low density lipoprotein (LDL) subfractions in patients with type 2 diabetes mellitus: baseline LDL profile determines specific mode of action.

The objective of this study was to determine the effect of slow-release (XL) fluvastatin on low density lipoprotein (LDL) subfractions in type 2 diabetes. A multicenter, double-blind, randomized, parallel-group comparison of fluvastatin XL 80 mg (n = 42) and placebo (n = 47), each given once-daily for 8 wk, in 89 patients with type 2 diabetes (HbA1c: 7.2 +/- 1.0%, LDL cholesterol (LDL-C): 3.4 +/- 0.7 mmol/liter, high density lipoprotein cholesterol: 1.1 +/- 0.3 mmol/liter, and triglycerides (TG): 2.4 +/- 1.4 mmol/liter). At baseline and on treatment, plasma lipoproteins were isolated and quantified. Eight weeks of fluvastatin treatment decreased total cholesterol (-23.0%, P < 0.001), LDL-C (-29%, P < 0.001) and TG (-18%, P < 0.001), compared with placebo. At baseline, there was a preponderance of dense LDL (dLDL) (apolipoprotein B in LDL-5 plus LDL-6 > 25 mg/dl) in 79% of patients, among whom fluvastatin decreased all LDL subfractions, reductions in dLDL being greatest (-28%, P = 0.001; cholesterol in dLDL -29%). In patients with low baseline dLDL (apolipoprotein B in LDL-5 plus LDL-6 </= 25 mg/dl), but a preponderance of buoyant LDL (LDL-1 through LDL-3), fluvastatin significantly decreased only these subfractions. Fluvastatin 80 mg XL, once daily, decreased total cholesterol and total LDL-C. In patients with atherogenic dLDL, absolute changes of dLDL were most pronounced, emphasizing the value of fluvastatin treatment in type 2 diabetes. The antiatherogenic potential of fluvastatin in type 2 diabetes may thus be greater than that expected from its effects on LDL-C and TG alone.

Aged↗

PubMed2002

STAT5 activation induced by diabetic LDL depends on LDL glycation and occurs via src kinase activity.

Advanced glycation end products (AGEs) have been implicated in the accelerated vascular injury occurring in diabetes. We recently reported that LDL prepared from type 2 diabetic patients (dm-LDL), but not normal LDL (n-LDL) triggered signal transducers and activators of transcription STAT5 activation and p21(waf) expression in endothelial cells (ECs). The aims of the present study were to investigate the role of LDL glycation in dm-LDL- mediated signals and to analyze the molecular mechanisms leading to STAT5 activation. We found that glycated LDL (gly-LDL) triggered STAT5 activation, the formation of a prolactin inducible element (PIE)-binding complex containing STAT5, and increased p21(waf) expression through the activation of the receptor for AGE (RAGE). We also demonstrated that dm-LDL and gly-LDL, but not n-LDL treatment induced the formation of a stable complex containing the activated STAT5 and RAGE. Moreover, gly-LDL triggered src but not JAK2 kinase activity. Pretreatment with the src kinase inhibitor PP1 abrogated both STAT5 activation and the expression of p21(waf) induced by gly-LDL. Consistently, gly-LDL failed to activate STAT5 in src(-/-) fibroblasts. Collectively, our results provide evidence for the role of glycation in dm-LDL-mediated effects and for a specific role of src kinase in STAT5-dependent p21(waf) expression.

Cells, Cultured↗

PubMed1988

Lipolysis of LDL with phospholipase A2 alters the expression of selected apoB-100 epitopes and the interaction of LDL with cells.

To assess the effects of perturbing the surface of low density lipoprotein (LDL) on the conformation of apoB-100, LDL (d 1.030-1.050 g/ml) isolated from normal subjects were treated with phospholipase A2 (PL-A2) for 0.5 to 15 min. The resulting P-LDL and concurrent control LDL (C-LDL) incubated without PL-A2 were isolated by gel permeation chromatography. Approximately 50% of LDL-phosphatidylcholine was hydrolyzed in 2 min and approximately 85% in 5 min. Lysophosphatidylcholine compounds (LPC) and free fatty acids (FFA) accumulated during lipolysis but most of the LPC and all of FFA could be removed by adding FFA-free albumin to the lipolysis mixtures. Immunoreactivities of P-LDL and C-LDL were evaluated in competitive radioimmunoassays, using a library of anti-human LDL monoclonal antibodies directed against the major regions of apoB-100 (the T4, T3, and T2 thrombin fragments). One epitope defined by monoclonal antibody 465B6C3 and localized near the carboxyl end of the apoB-100 molecule became less immunoreactive (ED 50s increased); three other epitopes on the T2 fragment near the LDL receptor recognition site and four epitopes localized towards the middle (T3) and amino terminal (T4) regions did not change. Altered immunoreactivities were not related to LPC and FFA contents. Thus, the conformation of apoB-100 was selectively altered by phospholipolysis. The interactions of P-LDL with cultured fibroblasts were grossly altered: P-LDL were bound nonspecifically to fibroblasts of both normal and homozygous familial hypercholesterolemic subjects and P-LDL were not degraded. LPC and FFA retained in LDL did not explain these alterations, nor did changes of epitope expression near the LDL receptor recognition site. It is likely that the apoB-100 aberrant cell interaction is due to loss of surface phospholipids and "uncovering" of core lipids that react nonspecifically with cell surface components.

Apolipoprotein B-100↗

PubMed1989

High carbohydrate fat-free diet modulates epitope expression of LDL-apoB-100 and interaction of LDL with human fibroblasts.

High carbohydrate diets are known to increase the concentration of very low density lipoprotein (VLDL) and to lower the concentrations of low density lipoprotein (LDL) and high density lipoprotein (HDL) in plasma. Such diets also alter lipoprotein compositions and metabolism. The aims of the present study were to assess in detail the effects of a virtually fat-free high carbohydrate (CHO) diet (CHO greater than 85% and fat less than 1% of calories) on various aspects of LDL. Thirteen healthy normolipidemic volunteers ate a basal "American" diet and the CHO diet for 7 days each in a forward or reverse sequence. Fasting blood samples were drawn at the ends of each study period and analyzed for lipoprotein lipid and apolipoprotein concentrations. Compositions of LDL particles isolated by ultracentrifugation were characterized chemically, LDL sizes were assessed by nondenaturing gradient electrophoresis on 2-16% gels, and association and degradation of LDL with normal human skin fibroblasts were quantified in cell cultures. Immunoreactivities of apoB in LDL were tested in solid phase competitive binding radioimmunoassays using five monoclonal anti-LDL antibodies that reacted with defined epitopes of apoB-100. The study diet produced consistent decreases of LDL cholesterol and apoB concentrations by 25% and 17%, respectively. LDL compositions were altered. Mean LDL triglycerides increased 3% to 4% of total LDL mass (P less than 0.004), and LDL particle sizes decreased (P less than 0.01). In radioimmunoassays that contained monoclonal antibody B1B3, an antibody that inhibits binding of LDL to the LDL receptor, the mean ED50 value for LDL protein was reduced from 3.75 to 2.66 micrograms (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2000

Upregulation of endothelial receptor for oxidized LDL (LOX-1) by oxidized LDL and implications in apoptosis of human coronary artery endothelial cells: evidence from use of antisense LOX-1 mRNA and chemical inhibitors.

A specific lectin-like endothelial receptor for oxidized low density lipoprotein (LOX-1), distinct from the scavenger receptor in monocytes/macrophages, has been identified and cloned. In this study, we examined the regulation of LOX-1 by oxidized low density lipoprotein (ox-LDL) and determined the role of LOX-1 in ox-LDL-induced apoptosis of cultured human coronary artery endothelial cells (HCAECs). Incubation of HCAECs with ox-LDL (40 microg/mL), but not native LDL, for 24 hours markedly increased LOX-1 expression (mRNA and protein). After 48 hours of preincubation of HCAECs with a specific antisense to LOX-1 mRNA (antisense LOX-1), ox-LDL-mediated upregulation of LOX-1 was suppressed (P<0.01). In contrast, treatment of HCAECs with sense LOX-1 had no effect. Ox-LDL also induced apoptosis (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA laddering) of HCAECs in a concentration- and time-dependent fashion. LOX-1 played an important role in ox-LDL-mediated apoptosis of HCAECs because antisense LOX-1 inhibited this effect of ox-LDL. Polyinosinic acid and carrageenan, 2 different chemical inhibitors of LOX-1, also decreased ox-LDL-mediated apoptosis of HCAECs. Nuclear factor (NF)-kappaB was markedly activated in ox-LDL-treated HCAECs. The critical role of NF-kappaB activation became evident in experiments with antisense LOX-1, which abolished ox-LDL-mediated NF-kappaB activation. In this process, an NF-kappaB inhibitor, caffeic acid phenethyl ester, also inhibited ox-LDL-mediated apoptosis of HCAECs. These findings indicate that ox-LDL upregulates its own endothelial receptor. Ox-LDL-induced apoptosis is mediated by the action of LOX-1. In this process, NF-kappaB activation may play an important role as a signal transduction mechanism.

Apoptosis↗

PubMed1998

alpha-tocopherol protects the peroxidative modification of LDL to be recognized by LDL receptors.

BACKGROUND: Peroxidatively modified low-density lipoprotein (LDL) may contribute to the atherosclerotic process; therefore, protecting LDL against peroxidation may reduce or retard the progression of atherosclerosis. We evaluated the effect of alpha-tocopherol on copper-catalyzed LDL peroxidative modification. METHODS: The protective effects of alpha-tocopherol on copper-catalyzed LDL peroxidative modification were examined by measurement of the concentration of lipid hydroperoxides in LDL and by the provision of LDL cholesterol to lymphocytes via the LDL receptor-mediated pathway. RESULTS: The measurement of concentration of lipid hydroperoxides in LDL showed that alpha-tocopherol inhibited the peroxidative modification of LDL. Also, alpha-tocopherol preserved the ability of LDL to be recognized by LDL receptors in peripheral blood lymphocytes to the same extent as native LDL. CONCLUSION: These findings indicate that alpha-tocopherol may protect LDL against peroxidative modification, maintaining its ability to act as a ligand for LDL receptors in vivo.

Adult↗

PubMed1993

Technetium-99m labelled LDL as a tracer for quantitative LDL scintigraphy. I. Tracer purification, in vitro and in vivo long-term stability, in vitro validation and biodistribution.

The goal of the present study was to optimize technetium-99m labelling of low-density lipoprotein (LDL) and to investigate the in vitro and in vivo properties of the tracer to determine whether its application for quantitative scintigraphy of hepatic LDL receptor activity is feasible. LDL labelled with iodine-125 by the iodine monochloride method was used as a reference tracer. Comparison of different assessments of radiochemical purity [trichloro-acetic acid precipitation (%ppTCA), paper chromatography, size-exclusion chromatography and chloroform-methanol extraction] exhibited %ppTCA to be superior as a parameter of tracer quality. In spite of a high radiochemical purity immediately after labelling, modifications of 99mTc labelling of LDL did not overcome the poor long-term stability of the tracer. Subsequent dialysis in phosphate buffer over about 3 h sufficiently increased the long-term stability in vitro and in vivo. The competitive recognition of dialysed 99mTc-LDL and 125I-LDL with native LDL by high-affinity binding sites was demonstrated in human hepatoma cells (HepG2) and human fibroblasts. Biodistribution data of simultaneously injected 99mTc-LDL and 125I-LDL in New Zealand White rabbits showed a high uptake of both tracers in tissues with high LDL receptor activity, yet 99mTc-LDL uptake exceeded 125I-LDL uptake by two- to sevenfold. In contrast to 125I-LDL, 99mTc-LDL showed a higher unspecific uptake into the bone marrow and the spleen, suggesting an additional uptake mechanism probably via the scavenger pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Dietary fat saturation in rhesus monkeys affects LDL concentrations by modulating the independent production of LDL apolipoprotein B.

In a recent study from this laboratory, rhesus monkeys fed a 90% palm oil/10% soybean oil-containing diet (PS), rich in 16:0 and 18:1 fatty acids, had decreased total and LDL cholesterol concentrations compared to monkeys fed a 90% coconut oil/10% soybean oil-containing diet (CS), rich in 12:0 and 14:0 fatty acids. To investigate the metabolic basis of these changes, homologous 125I-VLDL and 131I-LDL were injected simultaneously into eight monkeys (four per dietary group). Analysis of apo B specific activity curves revealed that PS monkeys had an increased pool size of VLDL apo B (P less than 0.02), a 3-fold increase in the total VLDL apo B transport rate (P less than 0.001), a decreased pool size of LDL apo B (P less than 0.01) and a 2-fold decrease in the total transport rate of LDL apo B (P less than 0.001), while the irreversible FCR for VLDL apo B and LDL apo B was similar between dietary groups. PS monkeys derived a greater percentage of LDL apo B from VLDL catabolism resulting in a greater transport rate of LDL apo B from VLDL catabolism (P less than 0.055), in comparison to CS monkeys. For CS monkeys the proportion as well as the amount of LDL apo B derived from VLDL-independent catabolism (i.e., LDL apo B derived from sources other than VLDL catabolism) was higher (P less than 0.001) than the values obtained in PS monkeys. In both dietary groups the proportion of VLDL apo B converted to LDL apo B was similar, although the absolute amount was higher for the PS monkeys (P less than 0.06). The proportion of VLDL apo B directly removed from the circulation was similar for both dietary groups, with the absolute amount being higher for the PS monkeys (P less than 0.001). Consistent with the lower pool size of LDL apo B and the higher pool size of VLDL apo B observed in PS monkeys, plasma and LDL cholesterol concentrations tended to be lower, whereas plasma triacylglycerol and VLDL cholesterol concentrations tended to be higher, but these changes were not statistically significant. Although total apo B and VLDL apo B transport rates were increased 2-3-fold in PS monkeys, LDL apo B concentration was reduced by 40% (P less than 0.02) attributed to a significant reduction in the mass and proportion of LDL apo B derived independent of VLDL catabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2006

Genotype of the mutant LDL receptor allele is associated with LDL particle size heterogeneity in familial hypercholesterolemia.

Small, dense LDL particles have been associated with an increased risk of coronary artery disease. In order to assess the potential contribution of the genotype of the LDL receptor to LDL particle size heterogeneity in familial hypercholesterolemia (FH), we examined the electrophoretic characteristics of LDL particles in a large cohort of FH heterozygotes and controls. A total of 259 FH heterozygotes and 208 controls participated in the study. FH subjects were carriers of one of the nine French Canadian mutations in the LDL receptor gene. LDL particles were characterized by polyacrylamide gradient gel electrophoresis following a 6-week lipid-lowering drug-free baseline period. LDL-peak particle diameter (LDL-PPD), representing the most abundant LDL particle subpopulation, was significantly smaller in FH heterozygotes carrying a negative-receptor mutation than in subjects carrying a defective-receptor mutation (negative-receptor = 257.3 +/- 4.1 A versus defective-receptor = 259.0 +/- 4.3 A, p = 0.0006). No significant difference in plasma CETP concentrations was found between these two genotypic groups. Moreover, compared with controls having low triglyceride levels, negative-receptor subjects with high triglyceride levels had a relative risk of 19.6 (p < 0.0001) of having small, dense LDL particles while this risk was not significantly increased among defective-receptor subjects. Multivariate analysis showed that the LDL receptor status accounted for 5.7% of the variance in the LDL-PPD after adjustment for covariates. These results suggest that the genotype of the mutant LDL receptor allele was independently associated with variations in LDL-PPD and could partly explain why negative-receptor FH heterozygotes may be at greater risk of cardiovascular disease than defective-receptor FH subjects.

Adult↗