LDL apheresis

Low density lipoprotein (LDL) apheresis provides a safe and effective means of treating patients with homozygous familial hypercholesterolaemia (FH). It also has a role in preventing the progression of coronary artery disease in heterozygotes and others with severe dyslipidaemia who are refractory to or intolerant of high doses of lipid-lowering drugs. Established methods involve either adsorption of apolipoprotein B-containing lipoproteins by affinity columns containing anti-apolipoprotein B antibodies or dextran sulphate, or their precipitation at low pH by heparin, in each instance after first separating plasma from blood cells with a cell separator. The most recently developed method enables lipoproteins to be adsorbed directly from whole blood, using polyacrylate columns. All 4 methods have proved to be similarly efficient when used weekly or biweekly to lower LDL cholesterol and Lp(a) without unduly reducing HDL cholesterol. Economic constraints restrict the use of LDL apheresis to the treatment of potentially fatal disorders such as FH, where there is clear evidence of benefit compared with conventional therapy. Widening the indications to include the treatment of other dyslipidaemic disorders such as steroid-resistant nephrotic syndrome, post-transplant donor vessel disease, stroke and prevention of re-stenosis after coronary angioplasty requires evidence from controlled trials that is currently lacking.     

Human coronary smooth muscle cells internalize versican-modified LDL through LDL receptor-related protein and LDL receptors

Versican-like proteoglycans are the main component of the intimal extracellular matrix interacting with low density lipoprotein (LDL). The aim of this study has been to investigate the receptors involved in versican-modified LDL uptake by human vascular smooth muscle cells (VSMCs). We have found that versican-LDL interaction leads to the following: (1) monomeric LDL particles that are similar in size and electrophoretic mobility to native LDL but that have a higher capacity to induce intracellular cholesteryl ester (CE) accumulation and (2) fused LDL particles similar in size to those obtained by vortexing. The precipitable fraction of versican-LDL, composed of 50% monomeric and 50% fused LDL particles, induced a dose-response increase in the CE content of VSMCs. Anti-LDL receptor antibody decreased the CE accumulation derived from monomeric LDL particles by 88 +/- 3% and that derived from the total precipitable fraction by 45 +/- 3%. Inhibition of LDL receptor-related protein expression by antisense oligodeoxynucleotides reduced the CE accumulation derived from the precipitable fraction by 65 +/- 2.8%, whereas it did not produce any effect on the CE accumulation derived from monomeric LDL. These results suggest that versican-LDL induces CE accumulation in human VSMCs by the LDL receptor (monomeric particles) and LDL receptor-related protein (fused LDL).     

Circulating autoantibodies to oxidized LDL correlate with arterial accumulation and depletion of oxidized LDL in LDL receptor-deficient mice

Autoantibodies to oxidized low density lipoprotein (OxLDL) are elevated in some human populations with increased risk of atherosclerosis. To determine whether autoantibody levels to epitopes of OxLDL reflect the extent of aortic atherosclerosis and the content of OxLDL, we measured IgG and IgM autoantibody titers to malondialdehyde (MDA)-LDL and copper-oxidized LDL (Cu-OxLDL) in 43 LDL receptor-deficient mice consuming atherogenic and regression diets. Antibody titers were correlated to percent atherosclerotic surface area, aortic weight, and aortic OxLDL content, measured as the in vivo uptake of (125)I-MDA2, a monoclonal antibody to MDA-LDL. All mice were fed an atherogenic diet for 6 months, and 1 group was euthanized. The other 3 groups were fed an atherogenic diet (fat/CHOL group), normal mouse chow (chow group), or mouse chow supplemented with vitamins E and C (chow+VIT group) for an additional 6 months. After dietary intervention, compared with their own baseline, autoantibody titers to MDA-LDL and Cu-OxLDL increased significantly in the fat/CHOL group, whereas they did not change or decreased significantly in the chow and chow+VIT groups. Aortic weight and surface area showed significant progression in the fat/CHOL group, mild progression in the chow group, and no progression in the chow+VIT group (P<0.001), whereas OxLDL content actually decreased in the latter 2 groups (P<0.001). Significant correlations were seen with MDA-LDL autoantibody titers and OxLDL content (IgM, R=0.64 and P=0.0009; IgG, R=0.52 and P=0.009), as well as with percent surface area and aortic weight. These data support the hypothesis that autoantibody titers to OxLDL reflect changes in OxLDL content in atherosclerotic lesions of LDL receptor-deficient mice. Whether autoantibody titers to OxLDL will provide similar valuable insights into the extent of human atherosclerosis, particularly anatomic measurements of plaque burden and OxLDL content, remains to be determined.     

Expression of LDL receptor and uptake of LDL in mouse preimplantation embryos

OBJECTIVES: The aim of this study is to address the role of low-density lipoprotein (LDL) on mouse preimplantation embryos. METHODS: The temporal expression of low-density lipoprotein receptor (LDLR) and side-chain cleavage cytochrome P450 (P450scc) mRNAs in mouse oocytes and preimplantation embryos up to the hatched blastocyst stage were analyzed by RT-PCR and nested PCR techniques. Simultaneously, the expression of LDLR in the protein level was analyzed by fluorescent immunohistochemistory at oocyte, 4-cell embryo and blastocyst. Uptake of LDL was analyzed using the LDL labeled with the fluorescent probe DiO. RESULTS: LDLR mRNA was detected at oocyte, 8-cell, morula, blastocyst and hatched blastocyst stages. P450scc mRNA was detected at oocyte, 1-cell, 2-cell, 4-cell, blastocyst and hatched blastocyst stages. LDLR protein was detected in blastocyst. P450scc protein was detected in oocyte, 4-cell, and blastocyst. LDL-DiO was taken into embryo at blastocyst stage and this uptake was competitively suppressed by excess unlabeled LDL. CONCLUSIONS: It is suggested from the present study that LDLR may play an essential role in uptake of exogenous LDL into blastocyst stage and cholesterol derived from LDL may be the source of steroid hormone synthesis.     

Low-density lipoprotein (LDL) oxidizability before and after LDL apheresis.

Oxidation of low-density lipoprotein (LDL) plays a major role in the development of atherosclerosis. Hypercholesterolemia has been associated with enhanced in vitro oxidation of LDL, and lipid-lowering therapy reduces LDL oxidizability. In the present study, we investigated whether LDL apheresis performed with different techniques affects in vitro diene formation (lag phase) and modification of apolipoprotein B-100 (apoB). Baseline and posttreatment diene formation was correlated with the baseline pattern of plasma total fatty acids. We then performed a computer-simulation study to test the hypothesis that LDL apheresis-induced changes in LDL oxidizability are related to changes in the mass ratio between freshly produced and older LDL. In 19 patients aged 49+/-7 years with heterozygous familial hypercholesterolemia (FH) regularly treated with either immunoadsorption, heparin-induced LDL precipitation (HELP), or dextran sulfate (DS) adsorption, we determined lipoprotein levels, the lag phase, apoB modification, and the fatty acid pattern in plasma samples drawn at the onset and termination of one LDL apheresis. LDL apheresis significantly decreased total cholesterol, high-density lipoprotein (HDL) cholesterol, LDL cholesterol, and triglycerides by 50.4%, 14.9%, 62.6%, and 33.6%, respectively. The lag phase increased by a significant mean of 9.8%; the charge of apoB was not altered. The lag phase before treatment positively correlated with the baseline concentration of plasma total palmitic, myristic, and oleic acid. The increase in the lag phase during treatment correlated with a high pretreatment concentration of lauric, linoleic, and docosahexanoic acid. The simulation study indicates that a temporary imbalance between two LDL compartments, one representing freshly secreted LDL and the other representing older LDL, could explain the observed increase in the lag phase after LDL apheresis. In conclusion, in patients with heterozygous FH, LDL apheresis performed with different techniques decreases the susceptibility of LDL to oxidation. This decrease may be related to a temporary mass imbalance between freshly produced and older LDL particles. Furthermore, the baseline fatty acid pattern influences pretreatment and posttreatment susceptibility to oxidation.     

Antibodies to oxidised LDL

In prospective studies antibodies to oxidised LDL (low density lipoprotein) have been shown to predict myocardial infarction and progression of carotid atherosclerosis in non-autoimmune subjects. The antibodies to oxidised LDL are crossreactive with antiphospholipid antibodies most likely due to their binding to oxidised phospholipids. The frequent occurrence of these antibodies and their association with arterial thrombosis in patients with SLE and antiphospholipid syndrome suggest their involvement in the development of accelerated atherosclerosis in these patients. Some in vitro studies suggest that antibodies to oxidised LDL may have an atherogenic effect by enhancing the lipid accumulation into macrophages in the atherosclerotic vessels. These antibodies can be considered as markers of the pathogenic determinants of atherosclerosis, such as enhanced lipid oxidation, proinflammatory stage and impaired vasodilatation.     

Colesevelam HCl reduces LDL particle number and increases LDL size in hypercholesterolemia

BACKGROUND: Although LDL-cholesterol (LDL-C) remains the target of cholesterol-lowering therapy, high levels of LDL particle numbers better differentiate cardiovascular risk than LDL-C concentrations. Colesevelam HCl, a novel bile acid sequestrant, has also been shown to reduce mean LDL-C by 18% with the maximum dose. The effect of colesevelam HCl on LDL particle number and particle size has not been previously published. LDL particle number and particle size were measured by NMR spectroscopy. METHODS: In this multi-center, double-blind, placebo-controlled study, 149 patients with moderate hypercholesterolemia (LDL-C > 160 mg/dL [4.14 mmol/L]; triglycerides < 300 mg/dL [3.39 mmol/L]) were randomized to colesevelam HCl (1.5-3.75 g/d) or placebo for 6 weeks. RESULTS: Colesevelam HCl reduced LDL particle number by 6.8% at a dosage of 3.0 g/d (P = 0.031), and 13.7% (P = 0.0002) at a dosage of 3.75 g/d. Baseline levels of triglycerides or LDL size did not influence changes in LDL particle number. Further, mean LDL particle size increased with colesevelam HCl 3.75 g/d (1.1% increase versus baseline, P < 0.05). CONCLUSION: For patients with moderate hypercholesterolemia, treatment with colesevelam HCl at the recommended starting dose of 3.75 g/d lowered LDL particle number, and increased mean LDL particle size.     

Mutation in EGFP domain of LDL receptor-related protein 6 impairs cellular LDL clearance

Mutation in the EGFP domain of LDL receptor-related protein 6 (LRP6(R611C)) is associated with hypercholesterolemia and early-onset atherosclerosis, but the mechanism by which it causes disease is not known. Cholesterol uptake was examined in cells from LRP6(+/-) mice and LRP6(R611C) mutation carriers. Splenic B cells of LRP6(+/-) mice have significantly lower LRP6 expression and low-density lipoprotein (LDL) uptake than those of the wild-type littermates. Although similar levels of total LRP6 were found in lymphoblastoid cells (LCLs) of LRP6(R611C) mutation carriers and those of the unaffected family member, LDL uptake was significantly lower in the mutant cells. Mutant and wild-type receptors show similar affinities for apolipoprotein B at neutral pH. LRP6 colocalized with LDL and was coimmunoprecipitated with NPC1 (Niemann-Pick disease type C1), an endocytic regulator of LDL trafficking. However, the cellular localization of LRP6 in the mutant cells shifted from cell surface to late endosomes/lysosomes. Plasma membrane expression levels of LRP6(R611C) was lower compared to wild-type receptor and declined to a greater extent in LDL-rich medium. Further examinations revealed lower efficacy of apolipoprotein B dissociation from LRP6(R611C) compared to wild-type receptor at an acidic pH. These studies identify LRP6 as a receptor for LDL endocytosis and imply that R611C mutation results in reduced LRP6 membrane expression and decreased LDL clearance. Based on our findings, we conclude that the increased affinity of the mutant receptor for LDL in acidic pH leads to their impaired dissociation in late endosomes, which compromises their recycling to the plasma membrane.     

Reversible and irreversible non-internalized LDL and methyl LDL accumulation by human fibroblasts.

In previous in vivo animal studies, we showed that low density lipoprotein (LDL) accumulated irreversibly at the edges of healing arterial lesions rather than being internalized and degraded. To see if similar LDL accumulation occurs in vitro, fibroblasts from normal and homozygous familial hypercholesterolemic (FH) subjects were incubated at 37 degrees C with 125I-LDL and 125I-methyl LDL; the latter is not recognized by any known LDL receptor. Normal fibroblast accumulation of LDL and methyl LDL (5 microg/ml) plateaued within 1 h at 200 and 100 ng/mg, respectively. With FH cells, both LDL and methyl LDL accumulation plateaued at 100 ng/mg. Lipoprotein accumulation by both cell types rose steeply at concentrations up to 15-25 microg/ml, and less so at higher concentrations. Except for degradation of LDL by normal cells, degradation was minimal, which indicated that much of the lipoprotein accumulation was unaccompanied by internalization. The accumulation of both lipoproteins by both cell types was greater at 37 degrees C than at 4 degrees C, and was inhibited between 43 and 75% by homologous unlabeled lipoprotein. To see if any accumulation was irreversible, cells were incubated with radiolabeled lipoproteins for 3 h (pulse), then with homologous unlabeled lipoproteins for up to 20 h (chase). About 50% of intact radiolabeled lipoprotein rapidly dissociated from cells into the medium in the first 4 h of the chase period. In contrast, between 4 and 20 h, most of the remaining intact LDL and methyl LDL appeared to be irreversibly bound, because it was released at a rate of only 0-1%/h. Thus, we conclude that, under the conditions studied, both reversible and irreversible non-internalized LDL binding play a major role in LDL accumulation by cultured cells.     

LDL receptor-related protein mediates uptake of aggregated LDL in human vascular smooth muscle cells

Foam cell formation is a key event in the onset and progression of atherosclerotic lesions. We have previously reported that internalization of aggregated low density lipoproteins (agLDLs) by vascular smooth muscle cells (VSMCs) produces cholesteryl ester (CE) accumulation in these cells. The aim of this study was to analyze whether the low density lipoprotein receptor-related protein (LRP) mediates the uptake of agLDL by VSMCs. First, immunocytochemistry and fluorescence microscopic analysis with the use of anti-LRP antibodies indicated that there was a high expression of LRP in VSMCs. Confocal microscopic analysis with the use of agLDLs labeled with fluorochrome 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine and anti-LRP antibodies showed the colocalization of agLDL and LRP. The second approach was to analyze the effect of LRP ligands on agLDL internalization; lactoferrin strongly inhibited CE accumulation from agLDLs (85.0+/-5.7% at 25 microg/mL) by impairing agLDL binding. Coincubation of agLDL with anti-LRP antibodies decreased in a dose-dependent manner agLDL-derived CE accumulation (from 20% at 12.5 microg/mL to 80% at 50 microg/mL). The third approach was to evaluate whether antisense LRP oligodeoxynucleotides were able to block agLDL internalization. Treatment of VSMCs with 5 micromol/L antisense LRP oligodeoxynucleotides reduced agLDL-derived CE accumulation by 84+/-2%. In conclusion, these results from immunologic, biochemical, and molecular interventions demonstrate that LRP mediates the binding and internalization of agLDL in human VSMCs. Because LRP is highly expressed in VSMCs and the uptake of 1 LDL aggregate amounts to the deposition of several hundreds of LDL particles, the uptake of agLDL through LRP could have a crucial role for lipid deposition in VSMCs.     

Treatment of small dense LDL

The increased frequency of small, dense LDL is associated with the risk of coronary heart disease (CHD). Possible mechanisms include the increased susceptibility of small, dense LDL to oxidation and its high affinity for LDL-receptor-independent cell surface binding sites. Although more than 30% of adult men in the USA have been reported to have small,dense LDL, only 5.4% of young Japanese men are affected. However, more than 76% of Japanese diabetics with coronary heart disease have small, dense LDL. Furthermore, almost half of all obese women (BMI > 35 kg/m(2)) have small, dense LDL. Our previous observation revealed that type 2 diabetics had smaller LDL even if they were apparently normolipidemic. In the normotriglycelidemic group there was also a close relationship between LDL size and plasma triglyceride. Diabetics with microalbuminuria had smaller LDL than those with normoalbuminuria, indicating the early nephrotoxicity of small, dense LDL. We also found that young men with high-normal blood pressure have smaller LDL than those with optimal blood pressure. Furthermore, LDL size was decreased not only in preeclamptic women but also in normal pregnant women. Finally, weight reduction by obese women through strict diet control, the treatment of diabetics by acarbose or troglitazone, and the treatment of hyperlipidemia by new statins as well as fibrates were all successful in increasing LDL size associated with decreased plasma triglyceride.     

The preparation of copper-oxidized LDL for the measurement of oxidized LDL antibodies by EIA

In the present study we try to define the optimal conditions for preparation of copper-oxidized low-density lipoprotein (oxLDL) to be used for the assay of oxLDL antibodies by enzyme immunoassay (EIA). Oxidation of LDL was monitored by measuring the formation of conjugated dienes at 234 nm and the generation of fluorescent products with emission at 430 nm when excitation is performed at 360 nm. The generation of immunogenic epitopes was evaluated by testing the reactivity of aliquots collected at different times during the oxidation process with human sera with high oxLDL antibody levels and with a purified human oxLDL antibody. The values of fluorescence emission at 430 nm correlated best with reactivity with oxLDL antibodies; strong reactivity was usually associated with values greater than 1.1 U. The time needed for fluorescence emission to reach maximum levels varied between 6 and 14 h for most LDL, but it was considerably longer in a few LDL preparations. The maximal reactivity of oxLDL with oxLDL antibodies was observed when the LDL oxidation reaction was stopped 4 or more hours after the fluorescence readings reached their peak. At this stage of the oxidation reaction, apolipoprotein B fragmentation and aggregation were observed as shown by Western blot analysis. The CV for 13 EIA runs of two reference oxLDL antibodies reacting with four different pools of standardized oxLDL prepared according to the stated guidelines was 14.5 and 3.9%, confirming the reproducibility of our oxidation conditions.