LDL protein nitration: implication for LDL protein unfolding

Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL⁻) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL⁻ assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC).The plasma LDL⁻ fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL⁻ revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the α-helical structures and β₂ sheet as well as cysteine oxidation to cysteic acid in β₁ sheet. Circular dichroism analyses showed that the α-helical content of LDL⁻ was substantially lower (∼25%) than that of native LDL (∼90%); conversely, LDL⁻ showed greater content of β-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO⁻) or SIN-1: similar amino acid modifications as well as conformational changes (loss of α-helical structure and gain in β-sheet structure) were observed. Both LDL⁻ and ONOO⁻-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO⁻-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R.It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.     

Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor

LPL mediates the uptake of lipoproteins into different cell types independent of its catalytic activity. The mechanism of this process and its physiological relevance are not clear. Taking into account the importance of the endothelial barrier for lipoprotein uptake, in vitro studies with primary aortic endothelial cells from wild-type and low density lipoprotein receptor (LDLR)-deficient (LDLR(-/-)) mice were performed. Addition of LPL almost doubled the uptake of LDL into wild-type cells. However, there was virtually no LPL-mediated change of LDL uptake into LDLR(-/-) cells. Upregulation of LDLR by lipoprotein-deficient serum/lovastatin in wild-type cells resulted in a 7-fold increase of LPL-mediated LDL uptake. Uptake of chylomicron remnants was not affected by LDLR expression. In proteoglycan-deficient cells, LPL did not increase the uptake of lipoproteins. The physiological relevance of this pathway was studied in mice that were both LDLR(-/-) and transgenic for catalytically inactive LPL in muscle. In the presence of LDLR, inactive LPL reduced LDL cholesterol significantly (13-24%). In the absence of LDLR, LDL cholesterol was not affected by transgenic LPL. Metabolic studies showed that in the presence of LDLR, LPL increased the muscular uptake of LDL by 77%. In the absence of LDLR, transgenic LPL did not augment LDL uptake. Chylomicron uptake was not affected by the LDLR genotype. We conclude that LPL-mediated cellular uptake of LDL, but not of chylomicrons, is dependent on the presence of both LDLR and proteoglycans.     

Minimally modified LDL is an oxidized LDL enriched with oxidized phosphatidylcholines

The oxidative modification of low-density lipoprotein (LDL) is involved in atherogenesis. Among a variety of modified LDLs mentioned in the literature, so-called minimally modified LDL (MM-LDL) was reported to have pro-atherogenic properties despite minimal changes in its oxidative measures. After treatment of LDL with 1 micro M FeSO(4) at 4 degrees C for 96 h, the resulting MM-LDL showed a slight increase in thiobarbituric acid-reactive substances (TBARS) and little association with macrophages. On the other hand, heavily oxidized LDL, which was prepared by copper-induced oxidation of LDL at 37 degrees C, showed a sharp increase in TBARS and strong association with macrophages. By introducing a fluorometric procedure to detect aldehyde-containing phosphatidylcholines (aldehyde-PCs), we examined the amounts of aldehyde-PCs in modified LDL preparations. Aldehyde-PCs increased to 23.4 pmol/ microg protein in MM-LDL, which was more than four-fold higher than in the heavily oxidized LDL. We conclude that MM-LDL is a unique type of oxidized LDL enriched with aldehyde-PCs.     

Cranberries inhibit LDL oxidation and induce LDL receptor expression in hepatocytes

Cardiovascular disease (CVD) is the leading cause of death in most industrialized countries. Cranberries were evaluated for their potential roles in dietary prevention of CVD. Cranberry extracts were found to have potent antioxidant capacity preventing in vitro LDL oxidation with increasing delay and suppression of LDL oxidation in a dose-dependent manner. The antioxidant activity of 100 g cranberries against LDL oxidation was equivalent to 1000 mg vitamin C or 3700 mg vitamin E. Cranberry extracts also significantly induced expression of hepatic LDL receptors and increased intracellular uptake of cholesterol in HepG2 cells in vitro in a dose-dependent manner. This suggests that cranberries could enhance clearance of excessive plasma cholesterol in circulation. We propose that additive or synergistic effects of phytochemicals in cranberries are responsible for the inhibition of LDL oxidation, the induced expression of LDL receptors, and the increased uptake of cholesterol in hepatocytes.     

Lipoprotein-X reduces LDL atherogenicity in primary biliary cirrhosis by preventing LDL oxidation

Hypercholesterolemic human LDL contains oxidized subfractions that have atherogenic properties. Paradoxically, atherosclerosis incidence is low in patients with primary biliary cirrhosis (PBC), a disease characterized by marked increases in plasma LDL, including the LDL subfraction lipoprotein-X (Lp-X). To investigate the mechanisms underlying this paradox, we first examined the propensity to oxidation of unfractionated LDL isolated from PBC patients. After prolonged incubation with copper, PBC-LDL failed to increase the oxidation index or electrophoretic mobility noted in control LDL. An admixture of PBC-LDL or Lp-X with control LDL prevented oxidation of the latter in a dose-dependent manner. PBC-LDL was also noncompetitive against copper-oxidized LDL (oxLDL) for binding with a murine monoclonal anti-oxLDL antibody in a competitive ELISA. OxLDL exerts its proapoptotic and antiangiogenic effects in part by inhibiting fibroblast growth factor 2 (FGF2) expression. Preincubation of oxLDL with PBC-LDL, but not control LDL, attenuated the inhibitory effects of oxLDL on FGF2 expression in cultured bovine aortic endothelial cells (ECs). The antioxidant and prosurvival properties of PBC-LDL diminished after the patients underwent orthotopic liver transplantation. These results suggest that Lp-X reduces LDL atherogenicity by preventing LDL oxidation to protect EC integrity in the presence of hypercholesterolemia. They also suggest that altering LDL composition may be as important as reducing LDL concentration in preventing or treating atherosclerosis.     

Modified methylenedioxyphenol analogs lower LDL cholesterol through induction of LDL receptor expression

Although statin therapy is a cornerstone of current low density lipoprotein (LDL)-lowering strategies, there is a need for additional therapies to incrementally lower plasma LDL cholesterol. In this study, we investigated the effect of several methylenedioxyphenol derivatives in regulating LDL cholesterol through induction of LDL receptor (LDLR). INV-403, a modified methylenedioxyphenol derivative, increased LDLR mRNA and protein expression in HepG2 cells in a dose- and time-dependent fashion. These effects were apparent even under conditions of HMG-CoA reductase inhibition. Electrophoresis migration shift assays demonstrated that INV-403 activates SREBP2 but not SREBP1c, with immunoblot analysis showing an increased expression of the mature form of SREBP2. Knockdown of SREBP2 reduced the effect of INV-403 on LDLR expression. The activation of SREBP2 by INV-403 is partly mediated by Akt/GSK3β pathways through inhibition of phosphorylation-dependent degradation by ubiquitin-proteosome pathway. Treatment of C57Bl/6j mice with INV-403 for two weeks increased hepatic SREBP2 levels (mature form) and upregulated LDLR with concomitant lowering of plasma LDL levels. Transient expression of a LDLR promoter-reporter construct, a SRE-mutant LDLR promoter construct, and a SRE-only construct in HepG2 cells revealed an effect predominantly through a SRE-dependent mechanism. INV-403 lowered plasma LDL cholesterol levels through LDLR upregulation. These results indicate a role for small molecule approaches other than statins for lowering LDL cholesterol.     

低密度脂蛋白受体生物化学

低密度脂蛋白受体广泛存在于机体内许多组织和细胞,在脂蛋白代谢中起了十分重要的作用。它是一种酸性糖蛋白,由839种氨基酸组成。根据它的结构特点及其与功能的关系,大致可以区分为5个不同的结构功能区。其中,较为重要的是发现第一、二功能区分别与受体结合特异性有关和与鼠EGF前体同源。     

LRP6 protein regulates low density lipoprotein (LDL) receptor-mediated LDL uptake

Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6(R611C) mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6(WT)) and LRP6(R611C) in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6(WT) was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6(WT) forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6(R611C). These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels.     

Baseline diene conjugation in LDL lipids: an indicator of circulating oxidized LDL

The wide acceptance of the diene conjugation-method in monitoring low-density lipoprotein (LDL) oxidation ex vivo has led to development of an assay, which measures the amount of baseline diene conjugation (BDC) in circulating LDL, and is an indicator of oxidized LDL in vivo. The LDL-BDC assay is based on precipitation of serum LDL with buffered heparin, and spectrophotometric determination of baseline level of conjugated dienes in lipids extracted from LDL. Compared to existing methods for oxidized LDL, LDL-BDC is fast and simple to perform. Chemical studies by HPLC and NMR have verified that LDL-BDC is a specific indicator of circulating mildly oxidized LDL. Validity of the assay is further indicated by strong correlation with the titer of autoantibodies against oxidized LDL. Clinical studies have shown that LDL-BDC is closely related to coronary, carotid, and brachial atherosclerosis. Moreover, several independent studies have demonstrated surprisingly strong associations between LDL-BDC and known atherosclerosis risk factors (obesity, physical inactivity, hypertension, diabetes, and arterial functions). Indeed, these studies seem to indicate that as an indicator of the risk of atherosclerosis LDL-BDC clearly exceeds sensitivity and specificity of the common lipid markers of atherosclerosis. It is concluded that LDL-BDC is a promising candidate in search for methods for the evaluation of in vivo LDL oxidation and the risk of atherosclerosis.     

阴离子型LDL吸附材料概述

动脉粥样硬化是导致心血管疾病发生的最重要因素。血液中低密度脂蛋白(LDL)浓度过高是引起动脉粥样硬化的主要原因。体外去除法是目前降低LDL浓度最有效的方法之一,吸附材料是影响LDL体外去除法降低LDL浓度效果的关键。阴离子型吸附材料是一种常用的吸附材料,因选材广泛、吸附效果佳备受关注,由发挥吸附功能的阴离子化合物配基和承载配基的载体基材组成,通过阴离子所带的负电荷与带正电的LDL产生特异性吸附。根据配基分子大小,阴离子型吸附材料主要分为大分子阴离子型和小分子阴离子型吸附材料,本文总结了国内外的阴离子型吸附材料主要研究现状及发展趋势。     

LDL receptor-related proteins in neurodevelopment

Low-density lipoprotein receptor-related proteins (LRPs) are evolutionarily ancient cell-surface receptors with diverse biological functions. All are expressed in the central nervous system and, for most receptors, animal models have shown that they are indispensable for successful neurodevelopment. The mechanisms by which they regulate the formation of the nervous system are varied and include the transduction of extracellular signals and the modulation of intracellular signal propagation, as well as cargo transport, the function most commonly attributed to this gene family. Here, we will summarize recent advances in our understanding of the molecular basis on which these receptors function during development .     

All three LDL receptor homology regions of the LDL receptor-related protein bind multiple ligands

The three complete human LDL receptor homology regions of the LDL receptor-related protein (sLRP2, sLRP3, and sLRP4) have been expressed in Pichia pastoris SMD1168 with constitutive coexpression of the receptor-associated protein (RAP). Each sLRP was purified to homogeneity after deglycosylation using a combination of anion-exchange and size exclusion chromatography. Mass spectrometry and N-terminal sequencing confirmed the identity of each fragment at purified yields of several milligrams per liter. Despite the large number of disulfide linkages and glycosylation sites in each LDL receptor homology region (sLRP), all were shown to be competent for binding to several LRP1 ligands. Each sLRP also bound human RAP, which is thought to be a generalized receptor antagonist, in solution-binding experiments. As expected, sLRP2 bound the receptor-binding domain of alpha(2)-macroglobulin (residues 1304-1451). All three sLRPs bound human apolipoprotein-enriched beta very low density lipoprotein, the canonical ligand for this receptor. All three sLRPs also bound lactoferrin and thrombin-protease nexin 1 complexes. Only sLRP4 bound thrombin-antithrombin III complexes. The results show that binding-competent LDL receptor homology regions (sLRPs) can be produced in high yield in P. pastoris and readily purified. Each sLRP has binding sites for multiple ligands, but not all ligand binding could be competed by RAP.     

In vivo metabolism of LDL subfractions in patients with heterozygous FH on statin therapy: rebound analysis of LDL subfractions after LDL apheresis

LDL can be subfractionated into buoyant (1.020-1.029 g/ml(-1)), intermediate (1.030-1.040 g/ml(-1)), and dense (1.041-1.066 g/ml(-1)) LDLs. We studied the rebound of these LDL-subfractions after LDL apheresis in seven patients with heterozygous familial hypercholesterolemia (FH) regularly treated by apheresis (58 +/- 9 years, LDL-cholesterol = 342 +/- 87 mg/dl(-1), triglycerides = 109 +/- 39 mg/dl(-1)) and high-dose statins. Apolipoprotein B (apoB) concentrations were measured in LDL subfractions immediately after and on days 1, 2, 3, 5, and 7 after apheresis. Compartmental models were developed to test three hypotheses: 1) that dense LDLs are derived from the delipidation of buoyant and intermediate LDLs (model A); 2) that dense LDLs are generated directly from LDL-precursors (model B); or 3) that a model combining both pathways (model C) is necessary to describe the metabolism of dense LDLs. In all models, it was assumed that apoB production and fractional catabolic rate (FCR) did not change with apheresis. Apheresis decreased buoyant, intermediate, and dense LDL-apoB by 60 +/- 12%, 67 +/- 5%, and 69 +/- 11%, respectively. Models B and C, but not model A, described the rebound data. The model with the greatest biological plausibility (model C) was used to estimate metabolic parameters. FCR was 1.05 +/- 0.86 d(-1), 0.48 +/- 0.11 d(-1), and 0.69 +/- 0.24 d(-1) for buoyant, intermediate, and dense LDLs, respectively. Dense LDL production was 17.3 +/- 0.2 mg/kg(-1)/d(-1), 58% of which was derived directly from LDL precursors (VLDL, IDL, or direct secretion), while 42% was derived from buoyant and intermediate LDLs. Thus, our data indicate that in statin-treated patients with heterozygous FH dense LDLs originate from two sources. Whether this is also valid in other metabolic situations (with predominant small, dense LDLs) remains to be determined.     

Immunological responses to oxidized LDL

Considerable evidence now points to an important role for the immune system in experimental models of atherosclerosis. We have reviewed the growing body of evidence that oxidation of LDL generates a wide variety of neoself determinants that lead to cellular and humoral immune responses. In particular, we have demonstrated that at least some of the oxidation-specific epitopes generated on the oxidized LDL particle, such as oxidized phospholipid epitopes, are also generated on apoptotic cells and are also present on the surface of some bacteria. Many of these same epitopes serve as important ligands mediating the binding and clearance of oxidatively damaged lipoprotein particles and apoptotic cells, and the innate immune response to these epitopes can be seen as a conserted response to effect their removal. In addition, other epitopes of OxLDL also undoubtedly play a role in the immune activation that characterizes the progressive atherosclerotic plaque. It will be of great importance to define the importance of the role of these responses and to understand which are beneficial and which deleterious. Such information could lead one day to novel therapeutic approaches to inhibit atherogenesis that take advantage of the ability to manipulate the immune response.     

ELISA measurement of LDL receptors

An enzyme-linked immunosorbent assay was developed for measurement of low density lipoprotein (LDL) receptors. A monospecific polyclonal antibody to LDL receptor purified from rat liver that reacted with rat, mouse, canine, and human LDL receptor was used. With this assay, LDL receptors could be measured on 2-4 x 10(5) adherent cells and 1.0 x 10(5) cells in suspension, although results were more variable with cell suspensions. Membranes from a variety of receptor-rich and receptor-poor tissues could be assayed directly after adherence of the membranes to the ELISA plate by an overnight incubation. In some instances, the quality of the assay was improved by first solubilizing the membranes. The sensitivity of the assay is such that between 0.15 and 2 micrograms of membrane protein is required. This could be obtained from leukocytes in a modest (20-30 ml) quantity of human blood. The assay was used to demonstrate the rapid down-regulation of LDL receptors in human mononuclear leukocytes in response to a cholesterol-containing meal. Overall, the results support the use of ELISA technology to measure LDL receptors, particularly for physiologic studies.     

Upregulation of hepatic LDL transport by n-3 fatty acids in LDL receptor knockout mice

We determined the effects of dietary n-6 and n-3 polyunsaturated fatty acids (PUFA) on parameters of plasma lipoprotein and hepatic lipid metabolism in LDL receptor (LDLr) knockout mice. Dietary n-3 PUFA decreased the rate of appearance and increased the hepatic clearance of IDL/LDL resulting in a marked decrease in the plasma concentration of these particles. Dietary n-3 PUFA increased the hepatic clearance of IDL/LDL through a mechanism that appears to involve apolipoprotein (apo)E but is independent of the LDLr, the LDLr related protein (LRP), the scavenger receptor B1, and the VLDLr. The decreased rate of appearance of IDL/VLDL in the plasma of animals fed n-3 PUFA could be attributed to a marked decrease in the plasma concentration of precursor VLDL. Decreased plasma VLDL concentrations were due in part to decreased hepatic secretion of VLDL triglyceride and cholesteryl esters, which in turn was associated with decreased concentrations of these lipids in liver. Decreased hepatic triglyceride concentrations in animals fed n-3 PUFA were due in part to suppression of fatty acid synthesis as a result of a decrease in sterol regulatory element binding protein-1 (SREBP-1) expression and processing. In conclusion, these studies indicate that n-3 PUFA can markedly decrease the plasma concentration of apoB-containing lipoproteins and enhance hepatic LDL clearance through a mechanism that does not involve the LDLr pathway or LRP.     

Native LDL and minimally oxidized LDL differentially regulate superoxide anion in vascular endothelium in situ

Low-density lipoprotein (LDL) and its oxidized derivatives are hypothesized to impair vascular function by increasing superoxide anion (O.). To investigate mechanisms in situ, isolated carotid arteries were incubated with native LDL (nLDL) or minimally oxidized LDL (mmLDL). With the use of en face fluorescent confocal microscopy and hydroethidine, an oxidant-sensitive fluorescent probe, we found that nLDL increased O. in vascular endothelium greater than fourfold by an N(omega)-nitro-L-arginine methyl ester (L-NAME)-inhibitable mechanism. In contrast, mmLDL increased O. in vascular endothelium greater than eightfold by mechanisms that were partially inhibited by L-NAME and allopurinol and essentially ablated by diphenyleneiodium. These data indicate that both nLDL and mmLDL uncouple endothelial nitric oxide synthase (eNOS) activity and that mmLDL also activates xanthine oxidase and NADPH oxidoreductase to induce greater increases in O. generation than nLDL. Western analysis revealed that both lipoproteins inhibited A-23187-stimulated association of heat shock protein 90 (HSP90) with eNOS without inhibiting phosphorylation of eNOS at serine-1179 (phospho-eNOS), an immunological index of electron flow through the enzyme. As HSP90 mediates the balance of.NO and O. generation by eNOS, these data provide new insight into the mechanisms by which oxidative stress, induced by nLDL and mmLDL, uncouple eNOS activity to increase endothelial O. generation.