LDL Receptor Function Research Articles

Atherogenic effect of Arecoline: A computational study.

There are over 600 million people worldwide covering Asian and Oceanic countries including India have the habit of chewing areca nut as masticator in different forms. Arecoline (C8H13NO2) has been reported as one of the abundant constituents of areca nut. A good number of scientific publications have made Arecoline responsible for oral cancer. Based on observation from clinical situation in North East India, one of the most betel quid chewing region of the country, we suspected a link between consumption of areca nut and Cerebro Vascular Disease like stroke. Therefore, we considered Low Density Lipoprotein (LDL) receptor as target and Arecoline as ligand and studied ligand –target interaction using computational tools. Also we considered High Density Lipoprotein (HDL) receptor as another target to see if Arecoline has any binding potential with it over and above LDL receptor. Docking result indicated that Arecoline and Cholesterol both, have affinity towards extracellular domain of Human LDL receptor but affinity of Arecoline is much higher (-12.3560.) than that of Cholesterol(-0.1810). Docking of Arecoline and 1, 2-Hexyl-1- cyclopentanone thiosemicarbazone (thiosemicarbazone) with Bovine HDL receptor showed that Arecoline also has the potential (Score, -6.2690Kcal/Mol) to block HDL receptor though its potential is less than that (score, -10.0509 Kcal/Mol) of control (thiosemicarbazone). We, therefore, suggest that by inhibiting endocytosis of LDL cholesterol because of blocking LDL receptor function and also by preventing LDL cholesterol uptake by liver from blood because of interference with HDL receptor, Arecoline may contribute to atherosclerosis. The study therefore, indicates a positive correlation between chewing of betel quid and Cerebro Vascular Disease.

Pharmacogenetic aspects in familial hypercholesterolemia with the special focus on FHMarburg (FH p.W556R)

ObjectiveFamilial hypercholesterolemia (FH) is an autosomal dominant inherited disorder caused by mutations in the low density lipoprotein receptor (LDLR) gene. FH is characterized by elevated plasma LDL cholesterol, premature atherosclerosis, and a high risk of premature myocardial infarction. In general, mutations within LDLR gene can cause five different classes of defects, namely: class I defect: no LDLR synthesis; class II defect: no LDLR transport; class III defect: no low density lipoprotein (LDL) to LDLR binding; class IV defect: no LDLR/LDL internalization; and class V defect: no LDLR recycling. One might expect that both the class of LDLR defect as well as the precise mutation influences the severity of hypercholesterolemia on one hand and the response on drug treatment on the other. To clarify this question we studied the effect of the LDLR mutation p.W556R in two heterozygote subjects.ResultsWe found that two heterozygote FH patients with the LDLR mutation p.W556R causing a class II LDLR defect (transport defective LDLR) respond exceedingly well to the treatment with simvastatin 40 mg/ezetimibe 10 mg. There was a LDL cholesterol decrease of 55 and 64%, respectively. In contrast, two affected homozygote p.W556R FH patients, in the mean time undergoing LDL apheresis, had no response to statin but a 15% LDL cholesterol decrease on ezetimibe monotherapy.ConclusionsThe LDLR mutation p.W556R is a frequent and severe class II defect for FH. The affected homozygote FH patients have a total loss of the functional LDLR and—as expected—do not respond on statin therapy and require LDL apheresis. In contrast, heterozygote FH patients with the same LDLR defect respond exceedingly well to standard lipid-lowering therapy, illustrating that the knowledge of the primary LDLR defect enables us to foresee the expected drug effects.

Mipomersen: evidence-based review of its potential in the treatment of homozygous and severe heterozygous familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal-dominant inherited disease with a prevalence of one in 500 (heterozygous) to one in 1,000,000 (homozygous). Mutations of the low-density lipoprotein (LDL) receptor gene, the apolipoprotein B100 gene, or the PCSK9 gene may be responsible for the disease. The resulting LDL hypercholesterolemia results in premature atherosclerosis as early as childhood (homozygous FH) or in adulthood (heterozygous FH). Current treatment modalities include lifestyle modification, combination drug therapy (statin-based), and apheresis. Mipomersen is an antisense oligonucleotide which inhibits apolipoprotein B production independent of LDL receptor function and thus works in homozygous FH, heterozygous FH, and other forms of hypercholesterolemia. Mipomersen is given 200 mg/week subcutaneously. Phase III studies indicate that the LDL cholesterol concentration can be reduced by 25%–47%, lipoprotein(a) levels by 20%–40%, and triglyceride concentrations by approximately 10%. In general, mipomersen has no effect on high-density lipoprotein cholesterol concentrations. Although there is considerable interindividual variability, the observed lipid effects are largely independent of age, gender, concomitant statin therapy, and underlying dyslipoproteinemia. The most common side effects are injection site reactions (70%–100%), flu-like symptoms (29%–46%), and elevated transaminases associated with an increased liver fat content (6%–15%). Mipomersen may be an interesting addon drug in patients with heterozygous or homozygous FH not reaching treatment goals, either because baseline values are very high or because high-dose statins are not tolerated.

Apolipoprotein E Mimetics and Cholesterol-Lowering Properties

Apolipoprotein E (apoE) is a ligand for clearance of lipoprotein remnants such as chylomicrons and very low-density lipoproteins. It has anti-atherogenic and anti-inflammatory properties. Therefore, there is extensive ongoing research to create peptides that can mimic properties of apoE. A number of synthetic peptides that encompass different regions of apoE have been studied for inhibiting inflammatory states, including Alzheimer disease. However, peptides that clear atherogenic lipoproteins, analogous to apoE, via enhanced hepatic uptake have not been previously reviewed. Toward this end, we describe the design and studies of a dual-domain apoE mimetic peptide, Ac-hE18A-NH(2). This peptide consists of residues 141-150, the putative receptor-binding region of human apoE, covalently linked to a well characterized class A amphipathic helix, 18A, which has no sequence homology to any other exchangeable apolipoprotein sequences. It demonstrates dramatic effects in reducing plasma cholesterol levels in dyslipidemic mouse and rabbit models. We discuss the scientific rationale and review the literature for the design and efficacy of the peptide. Analogous to apoE, this peptide bypasses the low-density lipoprotein receptor for the hepatic uptake of atherogenic lipoproteins via heparan sulfate proteoglycan (HSPG). ApoE mimetics such as Ac-hE18A-NH(2) may therefore restore or replace ligands in genetically induced hyperlipidemias to enable reduction in atherogenic lipoproteins via HSPG even in the absence of functional low-density lipoprotein receptors. Therefore, this and similar peptides may be useful in the treatment of dyslipidemic disorders such as familial hyperlipidemia and atherosclerosis.

An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations

The main causes of familial hypercholesterolemia (FH) are mutations in LDL receptor (LDLR) gene. Functional studies are necessary to demonstrate the LDLR function impairment caused by mutations and would be useful as a diagnostic tool if they allow discrimination between FH patients and controls. In order to identify the best method to detect LDLR activity, we compared continuous Epstein-Barr virus (EBV)-transformed B-lymphocytes and mitogen stimulated T-lymphocytes. In addition, we characterized both novel and known mutations in the LDLR gene. T-lymphocytes and EBV-transformed B-lymphocytes were obtained from peripheral blood of 24 FH patients and 24 control subjects. Functional assays were performed by incubation with fluorescent LDL followed by flow cytometry analysis. Residual LDLR activity was calculated normalizing fluorescence for the mean fluorescence of controls. With stimulated T-lymphocytes we obtained a better discrimination capacity between controls and FH patients compared with EBV-transformed B-lymphocytes as demonstrated by receiver operating characteristic (ROC) curve analysis (the areas under the curve are 1.000 and 0.984 respectively; P < 0.0001 both). The characterization of LDLR activity through T-lymphocytes is more simple and faster than the use of EBV-transformed B-lymphocytes and allows a complete discrimination between controls and FH patients. Therefore the evaluation of residual LDLR activity could be helpful not only for mutation characterization but also for diagnostic purposes.

Apolipoprotein Isoform E4 Does Not Increase Coronary Heart Disease Risk in Carriers of Low-Density Lipoprotein Receptor Mutations

In humans, the E4 allele of the apolipoprotein E gene is associated with increased coronary heart disease risk. Surprisingly, in rodents, apolipoprotein E4 only accelerates the atherosclerotic process when transgenic for the human low-density lipoprotein receptor (LDLR) protein. We therefore investigated whether the LDLR locus interacted with the apolipoprotein E gene genotype on coronary heart disease risk in patients clinically diagnosed with familial hypercholesterolemia with and without LDLR mutation. We investigated whether the presence of an LDLR mutation diminishing LDLR function was protective in E4/E4 carriers. In a cohort of 2400 patients clinically diagnosed with familial hypercholesterolemia, we found an LDLR gene mutation in 1383 patients, whereas in 1013 patients, such mutation was not present. In 92 patients homozygous for the apolipoprotein E4, the presence of an LDLR mutation conferred lower coronary heart disease risk (hazard ratio, 0.16; 95% CI, 0.05-0.58; P=0.005). Mirroring these results, the apolipoprotein E4/E4 genotype was also associated with lower coronary heart disease risk in patients with familial hypercholesterolemia with an LDLR mutation (hazard ratio, 0.26; hazard ratio, 0.08-0.80; P=0.02). LDLR function is key to the detrimental effects of apolipoprotein E4 in humans. Kinetic studies in humans are now required to study the consequences of our observation for prevention of both coronary heart disease and Alzheimer disease.

Mutation Analysis of the LDL Receptor Gene in Indian Families with Familial Hypercholesterolemia

Objective: Familial Hypercholesterolemia (FH) is a metabolic disorder inherited as an autosomal dominant trait characterized by an increased plasma low-density lipoprotein (LDL) level. The disease is caused by several different mutations in the LDL receptor (LDLR) gene. Several mutations have been reported in this gene in patients from several ethnic groups. Early identification of individuals carrying the defective gene could be useful in reducing the risk of atherosclerosis and myocardial infraction by the available therapeutic methods. The techniques available for determining the number of the functional LDLR molecules are difficult to carry out and expensive. Our study presents mutation analysis of the LDLR gene in 24 Indian families with FH. Material &amp; Methods: Peripheral blood samples were obtained form individuals after taking informed consent on the condition that each of these individuals had at least one first-degree relative affected with FH. Genomic DNA was isolated, exon-specific intronic primers were designed and used to amplify DNA samples from individuals.PCR products were directly subjected to automated DNA sequencing to detect the mutations. Along with the affected individuals, ten ethnically matched controls were also analyzed to determine the presence of the same mutations. Patients with Nephrotic Syndrome admitted to hospital were excluded from the study. Results: All the 24 patients had total cholesterol level above 300 mg/dl and LDL cholesterol level above 200mg/dl. Sequence analysis of the LDL receptor (LDLR) gene showed 3 novel mutations which have never been reported elsewhere. In exon 10 we reported g.29372_29373insC, which was found in all the 24 patients and was missence mutation coding for C (cysteine) instead of V (valine). Conclusion: Our study reported 3 novel mutations in 24 Indian families. These novel mutations are predicted to produce change in the amino acid and thus leading to the conformational changes in the structure of LDLR protein. Change in the LDLR protein makes the LDL receptor unable to transport the cholesterol in to the cell and hence cholesterol starts accumulating in the blood stream and leads to FH. Key Words: Familial Hypercholesterolemia; Mutation analysis; LDL Receptor geneDOI: http://dx.doi.org/10.3126/ajms.v2i2.4573Asian Journal of Medical Sciences 2 (2011) 82-86

Reduced penetrance of autosomal dominant hypercholesterolemia in a high percentage of families: Importance of genetic testing in the entire family

BackgroundAutosomal dominant hypercholesterolemias (ADHs) are characterised by increased plasma levels of total and LDL cholesterol, predisposing to premature atherosclerosis. ADHs comprise several diseases with undistinguishable phenotype, caused by mutations in different genes: LDLR, APOB and PCSK9. Genetic studies are usually performed in patients with altered cholesterol levels. However, some persons carrying pathogenic mutations are normocholesterolemic and there are no further studies about this subject. We have studied the frequency of families and individuals carrying ADH mutations who do not present the disease in Spanish population. MethodsWe have analysed genes known to cause ADH by direct sequencing in 24 ADH families (215 members). Functional effect of some LDLR gene mutations was assessed by transfecting cultured cells with plasmids. ResultsSix families with mutations presented 7 mutation carriers who did not show ADH phenotype: 30% of ADH families presented normocholesterolemic individuals, and 7% of carriers of pathogenic mutations did not show ADH phenotype. We have analysed the effect of some of these mutations and they are responsible for impaired LDL receptor function. We have excluded mutations in APOB and PCSK9 genes that could reduce LDLc levels. ConclusionsAn important percentage of ADH families presented individuals who do not show an ADH phenotype, but who are able to transmit the pathogenic mutation to their offspring. Genetic study of all subjects in ADH families should be performed in order to identify normocholesterolemic carriers that allow the detection of mutations in their descendants and the prevention of the disease consequences.

Role of an intramolecular contact on lipoprotein uptake by the LDL receptor

The LDL receptor (LDLR) is an endocytic receptor that plays a major role in the clearance of atherogenic lipoproteins from the circulation. During the endocytic process, the LDLR first binds lipoprotein at the cell surface and then traffics to endosomes, where the receptor releases bound lipoprotein. Release is acid-dependent and correlates with the formation of an intramolecular contact within the receptor. Human mutations at residues that form the contact are associated with familial hypercholesterolemia (FH) and the goal of the present study was to determine the role of contact residues on LDLR function. We show that mutations at nine contact residues reduce the ability of the LDLR to support lipoprotein uptake. Unexpectedly, only four of the mutations (W515A, W541A, H562Y and H586Y) impaired acid-dependent lipoprotein release. The remaining mutations decreased the lipoprotein-binding capacity of the LDLR through either reduction in the number of surface receptors (H190Y, K560W, H562Y and K582W) or reduction in the fraction of surface receptors that were competent to bind lipoprotein (W144A and W193A). We also examined three residues, distal to the contact, which were predicted to be necessary for the LDLR to adopt the acidic conformation. Of the three mutations we tested (G293S, F362A and G375S), one mutation (F362A) reduced lipoprotein uptake. Together, these data suggest that the intramolecular interface plays multiple roles in LDLR function.

Activation of LDL Receptor Expression by Small RNAs Complementary to a Noncoding Transcript that Overlaps the LDLR Promoter

Low-density lipoprotein receptor (LDLR) is a cell-surface receptor that plays a central role in regulating cholesterol levels. Increased levels of LDLR would lead to reduced cholesterol levels and contribute to strategies designed to treat hypercholesterolemia. We have previously shown that duplex RNAs complementary to transcription start sites can associate with noncoding transcripts and activate gene expression. Here we show that duplex RNAs complementary to the promoter of LDLR activate expression of LDLR and increase the display of LDLR on the surface of liver cells. Activation requires complementarity to the LDLR promoter and can be achieved by chemically modified duplex RNAs. Promoter-targeted duplex RNAs can overcome repression of LDLR expression by 25-hydroxycholesterol and do not interfere with activation of LDLR expression by lovastatin. These data demonstrate that small RNAs can activate LDLR expression and affect LDLR function.

Expression and regulation of a low‐density lipoprotein receptor exon 12 splice variant

As low-density lipoprotein receptor (LDLR) contributes to cholesterol and amyloid beta homeostasis, insights into LDLR regulation may facilitate our understanding of cardiovascular disease and Alzheimer's disease. Previously, we identified LDLR isoforms that lacked exon 12 or exons 11-12 and that are predicted to encode soluble, dominant negative, LDLR. Moreover, these isoforms were associated with rs688, an exon 12 polymorphism that was associated with LDL-cholesterol and Alzheimer's disease risk. In this study, we present evidence that although the truncated LDLR isoforms are translated in vitro, they represent < 0.1% of CSF proteins. As these LDLR isoforms likely represent a loss of mRNA-encoding functional LDLR, we then focused upon identifying intron-exon boundary and exonic splicing enhancer elements critical to splicing. Exon 12 inclusion is enhanced by altering the 5' splice site in intron 12 towards a consensus splice donor sequence, consistent with its being a weak 5' splice site. Additionally, of the nine evolutionarily conserved putative splicing enhancer regions within exon 12, two regions that flank rs688 were critical to exon 12 inclusion. Overall, these results suggest that LDLR splice variants represent a loss of mRNA encoding functional LDLR and provide insights into the regulatory elements critical for LDLR exon 12 splicing.

A Proprotein Convertase Subtilisin-like/Kexin Type 9 (PCSK9) C-terminal Domain Antibody Antigen-binding Fragment Inhibits PCSK9 Internalization and Restores Low Density Lipoprotein Uptake

PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.

Opposing Effects of Apolipoprotein M on Catabolism of Apolipoprotein B–Containing Lipoproteins and Atherosclerosis

Plasma apolipoprotein (apo)M is mainly associated with high-density lipoprotein (HDL). HDL-bound apoM is antiatherogenic in vitro. However, plasma apoM is not associated with coronary heart disease in humans, perhaps because of a positive correlation with plasma low-density lipoprotein (LDL). We explored putative links between apoM and very-low-density (VLDL)/LDL metabolism and the antiatherogenic potential of apoM in vivo. Plasma apoM was increased approximately 2.1 and approximately 1.5 fold in mice lacking LDL receptors (Ldlr(-/-)) and expressing dysfunctional LDL receptor-related protein 1 (Lrp1(n2/n2)), respectively, but was unaffected in apoE-deficient (ApoE(-/-)) mice. Thus, pathways controlling catabolism of VLDL and LDL affect plasma apoM. Overexpression (approximately 10-fold) of human apoM increased (50% to 70%) and apoM deficiency decreased ( approximately 25%) plasma VLDL/LDL cholesterol in Ldlr(-/-) mice, whereas apoM did not affect plasma VLDL/LDL in mice with intact LDL receptors. Moreover, plasma clearance of apoM-enriched VLDL/LDL was slower than that of control VLDL/LDL in mice lacking functional LDL receptors and LRP1, suggesting that apoM impairs the catabolism of VLDL/LDL that occurs independently of the LDL receptor and LRP1. ApoM overexpression decreased atherosclerosis in ApoE(-/-) (60%) and cholate/cholesterol-fed wild-type mice (70%). However, in Ldlr(-/-) mice the antiatherogenic effect of apoM was attenuated by its VLDL/LDL-raising effect. The data suggest that defect LDL receptor function leads to increased plasma apoM concentrations, which in turn, impairs the removal of VLDL/LDL from plasma. This mechanism opposes the otherwise antiatherogenic effect of apoM.

Cholesterol efflux via ATP-binding cassette transporter A1 (ABCA1) and cholesterol uptake via the LDL receptor influences cholesterol-induced impairment of beta cell function in mice

Cellular cholesterol accumulation is an emerging mechanism for beta cell dysfunction in type 2 diabetes. Absence of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) results in increased islet cholesterol and impaired insulin secretion, indicating that impaired cholesterol efflux leads to beta cell dysfunction. In this study, we aimed to determine the role of the LDL receptor (LDLr) in islet cholesterol uptake and to assess the contributions of cholesterol uptake compared with efflux to islet cholesterol levels. Islet cholesterol and beta cell function were assessed in mice lacking LDLr (Ldlr(-/-)), or apolipoprotein E (Apoe(-/-)), as well as in mice with beta-cell-specific deficiency of Abca1 crossed to Ldlr(-/-) mice. Hypercholesterolaemia resulted in increased islet cholesterol levels and decreased beta cell function in Apoe(-/-) mice but not in Ldlr(-/-) mice, suggesting that the LDL receptor is required for cholesterol uptake leading to cholesterol-induced beta cell dysfunction. Interestingly, when wild-type islets with functional LDL receptors were transplanted into diabetic, hypercholesterolaemic mice, islet graft function was normal compared with Ldlr(-/-) islets, suggesting that compensatory mechanisms can maintain islet cholesterol homeostasis in a hypercholesterolaemic environment. Indeed, transplanted wild-type islets had increased Abca1 expression. However, lack of the Ldlr did not protect Abca1(-/-) mice from islet cholesterol accumulation, suggesting that cholesterol efflux is the critical regulator of cholesterol levels in islets. Our data indicate that islet cholesterol levels and beta cell function are strongly influenced by LDLr-mediated uptake of cholesterol into beta cells. Cholesterol efflux mediated by ABCA1, however, can compensate in hypercholesterolaemia to regulate islet cholesterol levels in vivo.

Long-term Physiologically Regulated Expression of the Low-density Lipoprotein Receptor In Vivo Using Genomic DNA Mini-gene Constructs

Familial hypercholesterolemia (FH) is a condition caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Expression of LDLR is highly regulated and excess receptor expression is cytotoxic. To incorporate essential gene regulation into a gene therapy vector for FH, we generated vectors in which the expression of therapeutic human LDLR gene, or luciferase reporter gene, is driven by 10 kb of human LDLR genomic DNA encompassing the promoter region including elements essential for physiologically regulated expression. Using luciferase expression and specific LDL binding and internalization assays, we have shown in vitro that the genomic promoter element confers long-term, physiologically regulated gene expression and complementation of receptor deficiency in culture for 240 cell-generations. This was demonstrated in the presence of sterols or statins, modifiers of LDLR promoter activity. In vivo, we demonstrate efficient liver-specific delivery and expression of luciferase following hydrodynamic tail-vein injection and confirm that expression from the LDLR promoter element is sensitive to statin administration. We also demonstrate long-term LDLR expression from the 10-kb promoter element up to 9 months following delivery. The vector system that we describe provides the efficient delivery, long-term expression, and physiological regulation required for a successful gene therapy intervention for FH.

Mechanism of LDL binding and release probed by structure-based mutagenesis of the LDL receptor

The LDL receptor (LDL-R) mediates cholesterol metabolism in humans by binding and internalizing cholesterol transported by LDL. Several different molecular mechanisms have been proposed for the binding of LDL to LDL-R at neutral plasma pH and for its release at acidic endosomal pH. The crystal structure of LDL-R at acidic pH shows that the receptor folds back on itself in a closed form, obscuring parts of the ligand binding domain with the epidermal growth factor (EGF)-precursor homology domain. We have used a structure-based site-directed mutagenesis approach to examine 12 residues in the extracellular domain of LDL-R for their effect on LDL binding and release. Our studies show that the interface between the ligand binding domain and the EGF-precursor homology domain seen at acidic pH buries residues mediating both LDL binding and release. Our results are consistent with an alternative model of LDL-R whereby multiple modules of the extracellular domain interact with LDL at neutral pH, concurrently positioning key residues so that at acidic pH the LDL-R:LDL interactions become unfavorable, triggering release. After LDL release, the closed form of LDL-R may target its return to the cell surface.

Scavenger receptor, Class B, Type I provides an alternative means for β-VLDL uptake independent of the LDL receptor in tissue culture

Recent evidence suggests that scavenger receptor, class B, type I (SR-BI) plays a physiological role in VLDL metabolism. SR-BI was reported to mediate β-VLDL uptake; however, cellular details of this process are not well characterized. In the present study we show that SR-BI delivers cholesterol derived from β-VLDL to LDL receptor negative SR-BI over-expressing Chinese Hamster Ovarian cells (ldlA7-SRBI). Cell association of β-VLDL was ∼ 3 times higher after SR-BI over-expression, which was competed by β-VLDL, but only to a lesser extent by HDL and LDL. Almost all of the associated β-VLDL was located intracellularly, and therefore could not be released by a 50-fold excess of unlabeled β-VLDL. β-VLDL was degraded at a rate of 6 ng β-VLDL/mg cell protein and hour. In contrast to ldlA7 cells, β-VLDL association was competed by LDL in cells with a functional LDL receptor like CHO and HepG2 cells, indicating a strong impact of the LDL receptor in β-VLDL uptake. β-VLDL degradation was similar to ldlA7-SRBI cells. When β-VLDL uptake was followed using fluorescence microscopy, β-VLDL showed a different uptake pattern in SR-BI over-expressing cells, ldlA7-SRBI, compared to LDL receptor containing cells, CHO and HepG2.

Abstract: P847 THE PREVALENCE OF APOLIPOPROTEIN B-100 GENE MUTATIONS IN RUSSIAN FAMILIAL HYPERCHOLESTEROLEMIA PATIENTS

Familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB) are relatively common lipid disorders caused by mutations in the low-density lipoprotein receptor (LDLR) and apolipoprotein B (apo B) genes, respectively. Molecular analysis at these loci was performed in eight New Zealand subjects with clinical features of heterozygous FH. Utilization of an in vitro lymphocyte receptor assay demonstrated normal receptor function in four patients, three of whom screened positive for the founder-type apo B mutation, R3500Q, causing FDB. Four patients with reduced LDLR function, consistent with heterozygous FH, revealed three previously documented mutations in exons 3 (W66X), 6 (C292Y) and 7 (G322S) of the LDLR gene and, a novel 2-bp deletion (TC or CT) after nucleotide 1204 (or 1205) in exon 9. The remaining patient was found to be FH/FDB negative after extensive mutation screening using both denaturing gradient gel electrophoresis and heteroduplex-single strand conformation polymorphism analysis. Haplotype analysis at the LDLR and apo B loci finally excluded the likelihood that mutations in these two genes underlie the FH phenotype in the molecularly uncharacterized New Zealand family originating from the United Kingdom. This family represents a valuable source of material for future genetic dissection of autosomal dominant hypercholesterolemia (ADH), shown to be a heterogeneous disease through molecular analysis.

Remnant lipoproteins induced proliferation of human prostate cancer cell, PC-3 but not LNCaP, via low density lipoprotein receptor

Background: Hypertriglyceridemia has been shown to be one of the risk factors for prostate cancer. In this study, we investigated the effect of remnant lipoproteins on cell growth in prostate cancer cell lines. Methods: Remnant lipoproteins were isolated as remnant like particles (RLP) from human plasma. We used RLP for TG-rich lipoproteins and low density lipoproteins (LDL) for cholesterol-rich lipoproteins respectively and examined the effect of lipoproteins on proliferation of PC-3 and LNCaP cells using MTS assays. Moreover, we studied the effect of RLP and LDL treatment on the regulation of lipoprotein receptors in prostate cancer cells to investigate the relationship between lipoprotein-induced cell proliferation and lipoprotein receptor expression using real-time PCR, Western blotting assays and siRNA. Results: RLP effectively induced PC-3 cell proliferation more than LDL, whereas both RLP and LDL could not induce LNCaP cell proliferation except at a higher concentration of RLP. LDL receptor (LDLr) was expressed in both prostate cancer cells but there was a sharp difference of sterol regulation between two cells. In PC-3 cells, LDL decreased the LDLr expression in some degree, but RLP did not. Meanwhile LDLr expression in LNCaP was easily downregulated by RLP and LDL. Blocking LDLr function significantly inhibited both RLP- and LDL-induced PC-3 cell proliferation. Conclusions: This study demonstrated that RLP-induced PC-3 cell proliferation more than LDL; however, both RLP and LDL hardly induced LNCaP cell proliferation. The differences of proliferation by lipoproteins might be involved in the regulation of LDLr expression.

Disruption of SEMA4D Ameliorates Platelet Hypersensitivity in Dyslipidemia and Confers Protection Against the Development of Atherosclerosis

In dyslipidemic states, platelets become hyperreactive, secreting molecules that promote atherosclerosis. We have shown that the semaphorin family member, sema4D (CD100), is expressed on the surface of platelets and proposed that its role includes promoting thrombus growth by binding to nearby platelets and endothelial cells, both of which express sema4D receptors. Here we tested the hypothesis that deleting sema4D will attenuate the adverse consequences of dyslipidemia on platelets and the vessel wall. Platelet function and atherosclerotic lesion formation were measured in LDLR(-/-) and sema4D(-/-)LDLR(-/-) mice after 6 months on a high-fat diet. All of the mice developed the dyslipidemia expected on this diet in the absence of functional LDL receptors. However, when compared to LDLR(-/-) mice, sema4D(-/-) LDLR(-/-) mice had reduced lipid deposition in the descending aorta, a 6-fold decrease in the frequency of arterial occlusion and a reduction to near wild-type levels in the accumulation of platelets after injury. These differences were retained ex vivo, with a marked decrease in platelet accumulation on collagen under flow and in platelet aggregation. These results show that loss of sema4D expression reduces the platelet hyperactivity otherwise found in dyslipidemia, and confers protection against the development of atherosclerosis.