Homologous Low Density Lipoprotein Research Articles

Banana Prevents Plasma Oxidative Stress in Healthy Individuals

There is increasing evidence that lipid peroxidation and oxidative modification of low density lipoprotein (LDL) is important in atherogenesis. The present study was designed to study the effects of a single banana meal on plasma lipids and lipoprotein profile, plasma oxidative stress and susceptibility of LDL to oxidation in 20 healthy volunteers. Lipid and lipid peroxide (LPO) levels were measured before the meal (baseline, fasting) and 2 h after it (post-dose). The susceptibility to copper-induced oxidation of baseline and post-dose LDL was measured as conjugated diene (CD) formation. Results showed that the LPO contents in plasma, very low density lipoprotein (VLDL), LDL and high density lipoprotein (HDL) decreased significantly in the 2 h post-dose phase. Prolongation of lag phase and decrease of CD formation during LDL oxidation indicated that post-dose LDL was less susceptible to oxidative modification than the homologous fasting LDL. In conclusion, the consumption of banana reduces the plasma oxidative stress and enhances the resistance to oxidative modification of LDL.

Regulating Protective Immunity in Atherosclerosis

Atherosclerosis is a disease characterized by chronic inflammation. Looking back it is almost embarrassing how long it took for us to realize that the presence of these innate immune responses also was associated with activation of adaptive immunity. The first hints of an involvement of adaptive immunity in atherosclerosis came from studies performed by Goran Hansson and his coworkers about 20 years ago demonstrating the presence of activated T cells and expression of class II molecules in atherosclerotic plaques.1 This finding provoked an intense interest in the role of immunity in atherosclerosis, and it is now generally recognized that adaptive immune responses have a key role in determining the balance between disease progression and regression.2 It also focused attention on the immune system as a target for prevention and treatment of cardiovascular disease. Mice with defective adaptive immunity, such as SCID and Rag-1, develop less atherosclerosis indicating that adaptive immune responses are primarily proatherogenic. Oxidized LDL has been identified as one of the most important autoantigens in atherosclerosis.3 A large fraction of the T cells present in atherosclerotic plaques are specific for oxidized LDL,4 and oxidized LDL IgG is prevalent in the circulation of both humans and atherosclerosis-prone animals. However, studies evaluating the role of immune responses against oxidized LDL by immunizing animals with in vitro oxidized LDL unexpectedly revealed a protective effect.5,6 Although it still remains to be established whether naturally occurring autoimmune responses against oxidized LDL also have atheroprotective effects, these observations suggest the fascinating possibility of developing an immunomodulatory therapy for atherosclerosis. However, manipulating the immune system in complex diseases does not come without risk, and if the development of an immunomodulatory therapy for atherosclerosis is to become successful it will require detailed characterization of both disease-related and protective immune responses. The …

Increased Resistance to Oxidation of Betalain-enriched Human Low Density Lipoproteins

Betalains are natural pigments recently considered as compounds with potential antioxidative properties. In this work, ex vivo plasma spiking of pure either betanin or indicaxanthin, followed by isolation of low density lipoprotein (LDL), and measurement of its resistance to copper-induced oxidation, has been used to research if these betalains can bind to LDL and prevent oxidation of LDL lipids. When pooled human plasma from 10 healthy volunteers was incubated in the presence of 25–100 μM either betanin or indicaxanthin, incorporation of both compounds in LDL was observed, with a maximum binding of 0.52±0.08, and 0.51±0.06 nmoles of indicaxanthin and betanin, respectively, per mg LDL protein. Indicaxanthin-enriched and betanin-enriched LDL were more resistant than homologous native LDL to copper-induced oxidation, as assessed by the elongation of the induction period. The incorporated indicaxanthin, however, appeared twice as effective as betanin in increasing the length of the lag phase, while both compounds did not affect the propagation rate. Both betalains were consumed during the inhibition period of lipid oxidation, and delayed consumption of LDL-beta carotene. Indicaxanthin, but not betanin, prevented vitamin E consumption at the beginning of LDL oxidation, and prolonged the time of its utilization. The resistance of LDL to oxidation when vitamin E and indicaxanthin acted separately in a sequence, was lower than that measured when they were allowed to act in combination, indicating some synergistic interaction between the two molecules. No prooxidant effect over a large concentration range of either betanin or indicaxanthin was observed, when either betalain was added to the LDL system undergoing a copper-induced oxidation.These results show than indicaxanthin and betanin may bind to LDL, and are highly effective in preventing copper-induced lipid oxidation. Interaction with vitamin E appears to add a remarkable potential to indicaxanthin in the protection of LDL. Although molecular mechanisms remain uncompletely understood, various aspects of the action of betanin and indicaxanthin in preventing LDL lipid oxidation are discussed.

Concerted transcriptional activation of the low density lipoprotein receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: possible convergence of protein kinase a, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

Insulin and insulin-like growth factor I (IGF-I) can amplify gonadotropin-stimulated steroidogenesis by augmenting the expression of key sterol regulatory genes in ovarian cells, viz. low density lipoprotein (LDL) receptor, steroidogenic acute regulatory protein, and P450 cholesterol side-chain cleavage enzyme (CYP11A). The mechanisms underlying the foregoing bihormonal interactions are not known. Accordingly, in relation to the LDL receptor gene, the present study tests the hypothesis that insulin/IGF-I and LH can act via concerted transcriptional control of promoter expression. To this end, we transiently transfected primary monolayer cultures of porcine granulosa-luteal cells with a reporter vector containing the putative 5'-upstream full-length (pLDLR1076/luc) regulatory region (-1076 to +11 bp) of the homologous LDL receptor gene driving firefly luciferase in the presence or absence of insulin (or IGF-I) and/or LH (each 100 ng/ml). Combined exposure to LH and insulin (or IGF-I) stimulated LDL receptor transcriptional activity maximally at 4 h by 8- to 20-fold, as normalized by coexpression of Renilla luciferase. Further analysis of multiple 5'-nested deletional constructs of the LDL receptor gene promoter showed that deletion of -139 bp upstream of the transcriptional start site virtually abolished basal expression and promoter responsiveness to LH and insulin/IGF-I. In contrast, full basal activity and 60-80% of maximal monohormonal and bihormonal drive were retained by the -255 to +11 bp fragment. As LDL receptor gene expression in other tissues is negatively regulated by the abundance of intracellular free cholesterol, we assessed the impact of concomitant pretreatment of granulosa-luteal cells with an exogenous soluble sterol (25-hydroxycholesterol, 1 and 10 microM). Excess sterol markedly (50-70%) attenuated bihormonally and, in lesser measure, LH-stimulated and basal LDL receptor promoter expression, thus affirming a feedback-sensitive sterol-repressive region in this gene. Non-LH receptor-dependent agonists of protein kinase A (PKA), 8-bromo-cAMP (1 mM), and forskolin (10 microM) with or without insulin/IGF-I costimulation likewise augmented LDL receptor promoter expression with similar strong dependency on the -255 to -139 bp 5'-upstream region. To assess more specific PKA-dependent mediation of LH's contribution to combined hormonal drive, the LDL receptor (-1076 to +11 bp) reporter plasmid was cotransfected with a full-sequence rabbit muscle protein kinase inhibitor (PKI) minigene driven constitutively by a Rous sarcoma virus promoter. Expression of the latter PKA antagonist blocked transcriptional stimulation by LH alone as well as that by LH combined with insulin (or IGF-I) by 70-85% without reducing basal transcriptional activity. Transfection of a mutant inactive (Arg to Gly) Rous sarcoma virus/PKI gene confirmed the specificity of the PKI effect. To investigate the convergent role of the insulin/IGF-I effector pathway mediating bihormonal stimulation of LDL receptor promoter expression, transfected granulosa-luteal cells were pretreated for 30 min with two specific inhibitors of phophatidylinositol 3-kinase, wortmannin (100 nM) and LY 294002 (10 microM), or of mitogen-activated protein kinase kinase, PD 98059 (50 microM), U0126 (10 microM), or the latter's inactive derivative, U0124 (10 microM). Both classes of antagonists impeded the ability of insulin or IGF-I to enhance LH-stimulated LDL receptor promoter expression by 60-80%. In conclusion, the present analyses indicate that LH and insulin (or IGF-I) can up-regulate LDL receptor transcriptional activity supraadditively in porcine granulosa-luteal cells 1) via one or more agonistic cis-acting DNA regions located between -255 and -139 bp 5'- upstream of the transcriptional start site, 2) without abrogating sterol-sensitive repressive of this promoter, and 3) by way of intracellular mechanisms that include the PKA, phophatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

Variation of apolipoprotein B as a possible cause of decreased low density lipoprotein clearance and hypercholesterolemia

The fractional catabolic rate (FCR) for low density lipoprotein (LDL) apolipoprotein B (apo B) was studied to explore the variations in apo B as a possible cause of hypercholesterolemia. The FCR of radioiodine labelled autologous LDL and homologous LDL isolated from a normocholesterolemic subject were compared in forty-nine type II hypercholesterolemic males and females with the mean plasma concentration of total cholesterol of 7.78 mmol/l, LDL-cholesterol 5.41 mmol/l and triglycerides 2.09 mmol/l. In most patients the autologous LDL was catabolized at an equal rate and sometimes even faster than the homologous LDL. However, twelve out of forty-nine patients catabolized homologous LDL 0.8–19.3% faster than autologous LDL and several apo B polymorphisms were determined. No apo B-3500 or apo B-3531 mutations were detected. Patients with XbaI −/− (absence of cutting site) had lower total, IDL and LDL cholesterol and LDL apoB than the other genotypes. Patients with EcoRI +/+(presence of cutting site) had higher total, VLDL and LDL cholesterol and slower FCR for autologous LDL, and their VLDL was richer in cholesterol than that of patients with the EcoRI +/−. The MspI and ins/del polymorphisms were not associated with variations in the measured parameters. The apo E 4 was associated with higher VLDL and IDL cholesterol, higher triglycerides and LDL apo B than E 3/3. Overall, the determined apo B polymorphisms were not related to the slow clearance of autologous LDL among the 12 patients, in whom autologous LDL was cleared at a slower rate than homologous LDL. In conclusion, hypercholesterolemia can be due to particle-related slow clearance of LDL in some patients. However, this is not a common cause of hypercholesterolemia.

Immunization of LDL receptor-deficient mice with homologous malondialdehyde-modified and native LDL reduces progression of atherosclerosis by mechanisms other than induction of high titers of antibodies to oxidative neoepitopes.

We and others previously showed that immunization of rabbits with different forms of oxidized low density lipoprotein (LDL) significantly reduced atherogenesis. We now investigated the effect of continued immunization on atherosclerosis in LDL receptor-deficient (LDLR-/-) mice to determine whether a similar reduction of atherosclerosis occurred in murine models and whether this was due to humoral immune responses, ie, formation of high titers of antibodies to oxidation-specific epitopes. Three groups of LDLR-/- mice were repeatedly immunized with homologous malondialdehyde-modified LDL (MDA-LDL), native LDL, or phosphate-buffered saline (PBS) for 7 weeks. Extensive hypercholesterolemia and accelerated atherogenesis were then induced by feeding a cholesterol-rich diet for 17 weeks, during which immunizations were continued. Binding of immunoglobulin (Ig) M and IgG antibodies, as well as IgG1 and IgG2a isotypes, to several epitopes of oxidized LDL were followed throughout the study. After 24 weeks of intervention, atherosclerosis in the aortic origin was significantly reduced by 46.3% and 36.9% in mice immunized with MDA-LDL and native LDL, respectively, compared with PBS (133 558 and 157 141 versus 248 867 microm2 per section, respectively). However, the humoral immune response to oxidative neoepitopes in the MDA-LDL group was very different from that of the LDL or PBS group. IgG antibody binding to MDA-LDL and other epitopes of oxidized LDL, such as oxidized phospholipid (cardiolipin), oxidized cholesterol, or oxidized cholesteryl linoleate, but not native LDL, increased markedly in mice immunized with MDA-LDL, but not in mice immunized with native LDL or PBS. In the MDA-LDL group, both T helper cell (Th)2-dependent IgG1 antibody and Th1-dependent IgG2a antibody binding to oxidative neoepitopes increased significantly over time. The fact that mice immunized with both MDA-LDL and native LDL had a significant reduction in atherosclerosis, whereas only the MDA-LDL group developed very high titers of antibodies to oxidation-specific epitopes, suggests that the antiatherogenic effect of immunization is not primarily dependent on very high titers of antibodies to oxidation-specific epitopes but is more likely to result from the activation of cellular immune responses.

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.

Effects of stigmastanyl-phosphocholine (Ro 16-6532) and lovastatin on lipid and lipoprotein levels and lipoprotein metabolism in the hamster on different diets

Previous studies from our laboratory have shown that oral administration of stigmastanyl-phosphocholine (Ro 16-6532) reduces plasma cholesterol levels in experimental animals on diets free of added cholesterol. In the present study, effects of Ro 16-6532 and lovastatin on lipoprotein levels and metabolism were investigated in male golden Syrian hamsters. In hamsters fed a standard diet, Ro 16-6532 (1 mmol/kg/day) lowered cholesterol in all lipoprotein fractions, as well as apoB-100 and apoA-I. In contrast, lovastatin (25 mumol/kg/day) lowered high density lipoprotein (HDL)-cholesterol but had no effect on low density lipoprotein (LDL)-cholesterol or on apoB-100 or apoA-I while triglycerides and very low density lipoprotein (VLDL)-cholesterol increased. In hamsters fed a coconut fat-supplemented diet, Ro 16-6532 reduced all lipoproteins, with a stronger effect on VLDL- and LDL- than on HDL-cholesterol. Also apoB-100 was reduced. Lovastatin (50 mumol/kg/day) reduced LDL-cholesterol, HDL-cholesterol, and apoA-I while triglycerides and VLDL-cholesterol increased. The drop in LDL-cholesterol seen with both drugs in hamsters fed the diet supplemented with coconut fat occurred without any effect on the plasma removal rate of homologous LDL, or on the content of hepatic LDL-receptors. In contrast, the first phase of removal of homologous radioiodinated VLDL from plasma was markedly increased by both compounds, paralleled with an increased uptake of label in the liver and a decreased appearance of labeled apoB-100 in the LDL-fraction. Furthermore, retinyl ester-labeled chylomicrons were also cleared more rapidly in hamsters treated with Ro 16-6532. Hepatic uptake of label from VLDL and chylomicrons was strongly decreased by pre-injection of lactoferrin. In addition, Ro 16-6532 slightly decreased the secretion rate of VLDL in hamsters fed the coconut fat-supplemented diet. Taken together, these results indicate that the reduction of LDL-cholesterol after treatment with Ro 16-6532 and lovastatin observed in the hamster is mainly due to decreased conversion of VLDL into LDL, consequent to an increased hepatic removal of VLDL remnants. Ro 16-6532 also increased the liver uptake of chylomicron remnants. The hepatic uptake system implicated in this remnant removal can be completely blocked by lactoferrin. The nature of this uptake system is still unknown.

Oxidation of low density lipoprotein by mesangial cells may promote glomerular injury

Low density lipoprotein (LDL) deposition and local oxidation play a key role in the pathogenesis of atherosclerosis and may likewise contribute to glomerular injury. These studies were designed to determine whether cultured human mesangial cells oxidize homologous LDL and to compare the effects of unmodified and oxidized lipoprotein on cell proliferation, viability and eicosanoid production. Cell-mediated lipoprotein oxidation was demonstrated and could be suppressed by oxygen free radical scavengers and inhibitors of arachidonic acid metabolism. When incubated with cells, oxidized LDL (Ox-LDL) at concentrations up to and including 100 micrograms/ml reduced 3H-thymidine incorporation without causing cytotoxicity as assessed by lactate dehydrogenase release. Under the same conditions there was a concentration-dependent increase in the synthesis of prostaglandins E2,6-keto-PGF1 alpha and thromboxane B2. In contrast, unmodified LDL enhanced DNA synthesis at concentrations less than 40 micrograms/ml and had little effect on eicosanoid production. These results demonstrate that exogenous oxidized LDL inhibits mesangial cell proliferation and increases eicosanoid synthesis. Unmodified lipoprotein can be directly oxidized by these cells through mechanisms that involve generation of oxygen free radicals.

Low-density lipoprotein metabolism in rats treated with cyclosporine

Metabolic mechanisms underlying the observations of elevated cholesterol concentration of low-density lipoprotein (LDL) in organ-transplanted patients on long-term immunosuppressant cyclosporine therapy were explored using cyclosporine-treated rats as an experimental model. As in patients, treatment with cyclosporine induced a significant elevation of plasma cholesterol level, mainly in LDL cholesterol, with a decrease in high-density lipoprotein (HDL) cholesterol level. In an in vivo cross-over study design, differentially radioiodinated homologous LDL from donor cyclosporine-treated rats (Cyc-LDL) and excipient-only-treated control rats (Exc-LDL) were injected into recipient cyclosporine-treated rats (Cyc-rats), excipient-only-treated control rats (Exc-rats), and untreated rats (Unt-rats). From the isotope disappearance curves, the fractional catabolic rate (FCR) and production rate were calculated. The results showed that FCR and production rate were significantly reduced in Cyc-rats compared with control Exc-rats and Unt-rats. The decrease was independent of the donor LDL source. In vitro LDL ligand-receptor assays indicated a twofold higher degradation of Cycl-LDL by cultured rat fibroblasts, and hence could not account for the decreased clearance observed in vivo. These results suggest that the elevated concentrations of LDL cholesterol associated with cyclosporine treatment result not from a cyclosporine-induced modification of the LDL molecule, which could diminish its receptor-mediated clearance/catabolism, but possibly from an in vivo pharmacological property of cyclosporine such as an induced hepatic dysfunction.

Effects of individual and combined treatment with prostaglandins E2 and F2 alpha on progesterone secretion by ovine luteal cells supplemented with homologous serum lipoproteins in vitro.

Prostaglandin F2 alpha (PGF2 alpha) is a potent luteolysin, whereas prostaglandin E2 (PGE2) is generally luteotropic in vivo. To establish a model system for investigations of the mechanisms involved in these actions, we examined the effects of individual and combined treatment with PGE2 and PGF2 alpha on basal and ovine LH-stimulated progesterone secretion during long-term incubations conducted with and without supplemental homologous low-density lipoprotein (LDL) as substrate. Effects of both PGF2 and PGF2 alpha were concentration- and time-dependent and were further influenced by the presence of LDL and/or LH in medium. Neither of the prostaglandins exerted any significant effect before 48 h in culture, but distinctly different patterns of response to PGE2 and PGF2 alpha emerged thereafter. Low, but not high, concentrations of PGE2 increased progesterone secretion in the absence of LH, whereas PGF2 alpha (alone and in combination with PGE2) inhibited progesterone production in all medium formulations. The transcriptional inhibitor actinomycin effectively blocked the actions of PGF2 alpha, but had no effect on response to LH or PGE2. These data demonstrate that both the putative luteotropic actions of PGE2 and the potent, luteolytic effects of PGF2 alpha in vivo can be reproduced in long-term cultures of ovine luteal cells in vitro, and they suggest that the mechanism of PGF2 alpha-induced luteolysis may involve new protein synthesis.

Catabolism of low-density lipoprotein is altered in experimental chronic renal failure

Catabolism of low-density lipoprotein is altered in experimental chornic renal failure. In patients with chronic renal failure, cardiovascular disease accounts for a significant proportion of all deaths. Several factors contribute to the “accelerated” atherosclerosis in this population, including hyperlipidemia, the pathogenesis of which is multifactorial. We investigated low-density lipoprotein (LDL) metabolism in a remnant model of chronic renal failure in the guinea pig. After one and two-thirds nephrectomy, creatinine clearance decreased to one-sixth normal. Plasma cholesterol and triglyceride (TG) levels increased with induction of renal failure. Analysis of lipoprotein composition disclosed significant TG enrichment of both uremic very-low-density lipoprotein (VLDL) and uremic LDL compared with control lipoproteins. Plasma clearance of homologous LDL was evaluated in turnover studies in control and uremic guinea pigs. To discriminate between differences in catabolism related to the uremic lipoprotein particle and to the uremic host milieu, a crossover protocol was used comparing the fractional catabolic rate (FCR) after simultaneous injection into control and uremic animals of 125I-control LDL and 131I-uremic LDL. The FCR of native LDL was slower in uremic animals than in controls. In addition, FCR of uremic LDL was significantly less than that of control LDL in both groups. Degradation studies in cultured fibroblasts indicated substantially reduced degradation of uremic LDL compared with control LDL. These results suggest dual abnormalities of LDL catabolism in renal failure that are not only related to alterations in clearance mechanisms in the uremic environment, but also suggest important functional differences in the LDL particle itself isolated from uremic animals.

Correlations of plasma lipoproteins with LDL subfractions by particle size in men and women.

Nondenaturing gradient gel electrophoresis of plasma low density lipoprotein (LDL) has been used to identify major LDL subclasses that are influenced by genetic and other factors. In the present paper, this technique has been extended by measuring absorbance of lipid- or protein-stained gels as an index of concentration at intervals of 0.05 nm across the entire LDL particle size range (21.8-30 nm) in moderately overweight men (n = 115) and women (n = 78). When LDL absorbance levels were correlated with other lipoprotein variables, we found that the strengths of the correlations with each of triglycerides, apolipoprotein (apo) B, high density lipoprotein (HDL)2, and apoA-I achieve relative maximum values for two regions within the small LDL range (21-26 nm), one within LDL-IVB (22-23.2 nm) and a second within LDL-III (24.2-25.5 nm). We also found that the increase in LDL accompanying higher triglyceride levels occurs below 25.5 nm in men and between 24.5 and 26.5 nm in women, suggesting either that triglycerides are related to different LDL subclasses in men and women, or that particle sizes of metabolically homologous LDL subclasses may differ in men and women. As compared to analytic ultracentrifuge measurements, gradient gel measurements of LDL absorbance by the procedure described here provide greater resolution of LDL subclasses but less precision in estimating LDL levels.

Effect of prostaglandin E 1 on low density lipoprotein apo-B-receptor binding in human, rat and swine liver in vitro

The effect of PGE 1 on low density lipoprotein (LDL) apo-B-receptor binding was examined in huna, rat and swine liver. Autologous LDL (for humans and swines) and homologous LDL (for rats) were isolated by ultracentrifugation and labelled with 123I using Iodogen followed by purification with dialysis. LDL-concentrations of 0.1–6 μg protein/ml were used for direct binding assays investigating the specific binding of labelled LDL in presence of increasing PGE 1-concentrations (100 pM to 100 μM). In separate experiments the effect of PGE 1 on displacement of specifically bound 123I-LDL by unlabelled ones was studied. The binding capacities estimated by Scatchard analysis were similar for human and rat liver LDL-apo-B-receptor binding, however, swine liver exhibited a significantly (p<0.001) lower binding capacity for 123I-LDL. PGE 1 significantly (p<0.01-0.001) increased the amount of 123I-LDL specifically bound to the liver apo-B-receptors and th binding affinity in all liver preparations of the 3 species in a dose-dependent manner. PGE 1 also significantly increased competition of unlabelled LDL for 123I-LDL bound to specific apo-B-receptors in a dose-dependent manner (p<0.01–0.001) with an ED 50 of 123±64 nM for human liver, 90±102 nM for rat liver obtained during anasthesia, 74±23 nM for rat liver obtained after decapotation and 941±121 nM for swine liver. In human liver iloprost (ED 50=876±53 nM) and PGI 2 (ED 50=52±12 nM) were less effective than PGE 1, PGE 2 had no effect on LDL-induced competition. It is concluded that PGE 1 renders LDL more sensitive for apo-B-receptor binding suggesting a potential hypolipidemic action of PGE 1.

Aortic and coronary atheromatosis in a woman with severe hypercholesterolaemia without LDL receptor alterations

Familial hypercholesterolaemia (FH) is a monogenic disorder, with a strong family history, characterized by a deficiency in functional receptors for low density lipoproteins (LDL). The case of a patient with all the clinical traits of FH, including elevated cholesterol, xanthomas and early coronary and peripheral arterial lesions, but with a normal LDL receptor function, is described. In the patient the molecular weight and immunological properties of apolipoprotein (apo) B were normal; furthermore, autoantibodies to either LDL or to their receptor were also absent. The increased apo B/cholesterol ratio in LDL was compatible with the diagnosis of hyperapobetalipoproteinaemia. With the help of a turnover study using 131I homologous and 125I autologous LDL, it could be established that the patient had an almost three-fold increase in LDL-apo B biosynthesis, with, however, a fractional catabolic rate within normal limits. These findings pointed to the possibility of a genomic alteration in the region responsible for the control of apo B biosynthesis. However, extensive studies both at the cDNA level and in the 5' region of the apo B gene, failed to detect any significant alteration vs published nucleotide sequences. Although the exact mechanism for this unusual clinical presentation of an FH-like syndrome could not be uncovered, this case provides an extreme example of hypercholesterolaemia, with early and severe arterial disease, solely explained by an increased LDL biosynthesis.

Increased degradation of low density lipoproteins by mononuclear leukocytes associated with coronary artery disease

Low density lipoprotein (LDL) and peripheral blood mononuclear leukocytes (MNL) were isolated from patients with ( n = 11) and without ( n = 11) angiographically documented coronary artery disease (CAD). LDL degradation rates in MNL were determined in vitro using both autologous and homologous LDL. The mean rate of LDL degradation was 1.7-fold higher in CAD-MNL than in control-MNL ( P < 0.05), independent of the LDL source. The increased LDL degradation rate in CAD-MNL appeared to be due to an increased receptor-mediated LDL degradation rate in CAD-MNL and not to an increased CAD-LDL interaction with the receptor since LDL isolated from patients with and without CAD had similar in vitro degradation rates in HL-60 cells and 1,25-dihydroxyvitamin D 3-induced HL-60 macrophages. An increased ratio of apo B to cholesterol, specifically apo B to cholesteryl ester, was observed in LDL isolated from patients with CAD. LDL particles isolated from CAD patients contained 14.8% less cholesteryl ester than LDL from control subjects ( P < 0.01). The data suggest that CAD patients have an increased plasma LDL particle number even though they have similar plasma LDL-cholesterol levels as compared to control subjects. These data indicate that CAD patients with normal plasma LDL cholesterol levels have two metabolic abnormalities: an altered LDL composition resulting in particles with reduced cholesteryl ester content and an increased LDL catabolism resulting in an increased influx of LDL cholesterol into MNL; both of which may play a role in the development of coronary heart disease.

Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein.

Increasing evidence indicates that low density lipoprotein (LDL) has to be modified to induce foam cell formation. One such modification, oxidation of LDL, generates a number of highly reactive short chain-length aldehydic fragments of oxidized fatty acids capable of conjugating with lysine residues of apoprotein B. By immunizing animals with homologous malondialdehyde-modified LDL (MDA-LDL), 4-hydroxynonenal-LDL (4-HNE-LDL), and Cu+(+)-oxidized LDL, we developed polyvalent and monoclonal antibodies against three epitopes found in oxidatively modified LDL. The present article characterizes an antiserum and monoclonal antibody (MAL-2 and MDA2, respectively) specific for MDA-lysine, and an antiserum and monoclonal antibody (HNE-6 and NA59, respectively) specific for 4-HNE-lysine. In addition, a monoclonal antibody (OLF4-3C10) was developed against an as yet undefined epitope generated during Cu++ oxidation of LDL. With these antibodies, we demonstrated that MDA-lysine and 4-HNE-lysine adducts develop on apo-lipoprotein B during copper-induced oxidation of LDL in vitro. The application of these antibodies for immunocytochemical demonstration of oxidized lipoproteins in atherosclerotic lesions of progressive severity is described in the companion article. These antibodies should prove useful in studying the role of oxidatively modified lipoproteins as well as other oxidatively modified proteins in atherogenesis.

Low-density-lipoprotein receptors in different rabbit liver cells

Receptor-dependent uptake mechanisms for low-density lipoprotein (LDL) were studied in rabbit liver parenchymal and non-parenchymal cells. Hybridization studies with a cDNA probe revealed that mRNA for the apo (apolipoprotein) B,E receptor was present in endothelial and Kupffer cells as well as in parenchymal cells. By ligand-blotting experiments we showed that apo B,E-receptor protein was present in both parenchymal and non-parenchymal cells. Studies of binding of homologous LDL in cultured rabbit parenchymal cells suggested that about 63% of the specific LDL binding was mediated via the apo B,E receptor. Approx. 47% of the specific LDL binding was dependent on Ca2+, suggesting that specific Ca2+-dependent as well as Ca2+-independent LDL-binding sites exist in liver parenchymal cells. Methylated LDL bound to the parenchymal cells in a saturable manner. Taken together, our results showed that apo B,E receptors are present in rabbit liver endothelial and Kupffer cells as well as in the parenchymal cells, and that an additional saturable binding activity for LDL may exist on rabbit liver parenchymal cells. This binding activity was not inhibited by EGTA or reductive methylation of lysine residues in apo B. LDL degradation in parenchymal cells was mainly mediated via the apo B,E receptor.

Studies on mechanisms for enhanced clearance of low-density lipoproteins in patients with primary hypertriglyceridaemia.

Patients with primary hypertriglyceridaemia usually have increased clearance rates for plasma low-density lipoproteins (LDL). To evaluate the mechanisms for this effect, simultaneous turnover rates for autologous and normal homologous LDL were determined in 12 patients with primary hypertriglyceridaemia. On average, the autologous LDL was cleared more rapidly than the normal homologous LDL. Fractional catabolic rates (FCRs) for autologous LDL averaged 0.61 +/- 0.06 (SEM) pools d-1, whereas FCRs for homologous LDL averaged 0.49 +/- 0.04 pools d-1. In eight of the 12 patients the FCRs for 'hypertriglyceridaemic' LDL were found to be significantly higher than for normal LDL; in four others both forms of LDL were cleared at essentially the same rate. In all cases, however, both for the normal and 'hypertriglyceridaemic' LDL, clearance rates were higher than normal. Thus, besides the variability in LDL affinity for removal pathways, hypertriglyceridaemic patients appear to have an increased availability of LDL receptors for removal of circulating LDL.

Receptor-mediated uptake of homologous low-density lipoproteins by isolated liver parenchymal cells of fetal rats.

The binding and uptake of gold-labeled homologous, apolipoprotein E-free low-density lipoproteins (LDL) by isolated fetal rat liver parenchymal cells in suspension were studied ultrastructurally and morphometrically. Binding experiments using 125I-labeled LDL were also performed. After a 2-h preincubation in a lipoprotein-free medium and a subsequent 1-h postincubation in the presence of LDL-gold, fetal liver parenchymal cells exhibit a binding of 248 +/- 17 gold conjugates/100 micron plasma membrane and an uptake of 235 +/- 17 gold conjugates/100 micron2 cytoplasm. Compared with values obtained from freshly isolated non-preincubated cells, these data correspond to a 15-fold and an 18-fold increase in total binding and uptake of LDL-gold, respectively. Competition experiments reveal that this increase is mainly a result of a 23-fold stimulation of specific binding and a 44-fold stimulation of receptor-mediated uptake of LDL-gold. The 125I-LDL binding experiments give a Kd value of 6.3 X 10(-8) M and a maximum binding capacity of 17.3 fmol LDL/10(6) cells. Our data provide evidence, further to our in vivo studies, that fetal rat liver parenchymal cells possess high-affinity binding sites for native homologous apolipoprotein E-free LDL. These sites may correspond to B, E receptors of adult rat liver parenchymal cells.