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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2507-2514.)
© 1997 American Heart Association, Inc.

Articles

Interaction of Very-Low-Density, Intermediate-Density, and Low-Density Lipoproteins With Human Arterial Wall Proteoglycans

V. Anber; J. S. Millar; M. McConnell; J. Shepherd; ; C. J. Packard

From the University Department of Pathological Biochemistry, Glasgow Royal Infirmary, Glasgow G4 OSF, UK.

Correspondence to Dr Vian Anber, Department of Pathological Biochemistry, Glasgow Royal Infirmary, Alexandra Parade, Glasgow G4 OSF, UK.

	Abstract

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Abstract The specific interaction of lipoproteins with arterial wall constituents, particularly proteoglycans (APG), is believed to play an important role in the development of atherosclerosis. The objective of this study was to examine the interaction of apolipoprotein B (apoB) containing lipoprotein subfractions (VLDL1, S_f 60 to 400; VLDL2, S_f 20 to 60; IDL1, S_f 16 to 20; IDL2, S_f 12 to 16; LDLA, S_f 8 to 12; and LDLB, S_f 0 to 8) prepared by cumulative density gradient centrifugation with chondroitin sulfate-rich APG. Eighteen subjects were studied, and a similar pattern of interaction between the lipoprotein species and APG was found in all. The order of reactivity (as measured by increased turbidity due to insoluble complex formation) was IDL S_f 12 to 16 LDL S_f 8 to 12 > LDL S_f 0 to 8 > IDL S_f 16 to 20 >> VLDL S_f 20 to 60 > VLDL S_f 60 to 400. When the subjects were divided on the basis of their LDL subfraction profile, the extent of insoluble complex formation was highest in the group in which small, dense LDLIII was predominant; intermediate in the group whose LDL was mainly LDLII; and lowest in the group with a high proportion of LDLI (the mean reactivity, AU at 600 nm, of APG with IDL S_f 12 to 16 and LDL S_f 8 to 12 was 0.66; 0.62 and 0.46, 0.43 and 0.20, and 0.21 for the three groups, respectively). Fibrate lipid-lowering treatment decreased the percentage of LDLIII and increased the percentage of LDLI within total LDL and reduced the reactivity of all apoB-containing lipoprotein fractions toward APG. Sialic acid content varied in different lipoprotein subfractions, being the highest in VLDL and lowest in LDL. However, across lipoprotein species, it did not significantly correlate with APG-binding reactivity, suggesting that other factors are important in determining the interaction of lipoproteins with APG. Modification of LDL arginine and lysine residues abolished the ability of the lipoprotein to interact with APG, a finding that supports the hypothesis that the interaction is dependent on key positively charged amino acids on apoB. These findings demonstrate that (1) the overall reactivity of apoB-containing lipoproteins is greatest in individuals with small, dense LDL and (2) within an individual, IDL of S_f 12 to 16 is the most reactive species, and this may in part explain the positive correlation between IDL and risk of coronary heart disease.

Key Words: atherogenic lipoprotein phenotype • LDL subfractions • sialic acid • ciprofibrate

	Introduction

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Most investigators now agree that one or more apoB-containing lipoproteins, either in their native form or modified by oxidation or complex formation, are responsible for cholesterol deposition in the artery. The atherogenic role of low-density lipoprotein, especially small, dense LDL, is well established by epidemiological studies, clinical findings, and controlled trials.^1–3 One possible mechanism by which LDL can cause atherosclerosis is its interaction with arterial wall proteoglycans (APG). APG, especially chondroitin sulfate proteoglycans (CS-PG), have long been known to interact specifically with apoB-containing lipoproteins, mainly LDL, Lp(a), and to a lesser extent VLDL, but not with HDL.^4–9 This interaction leads to lipoprotein entrapment, modification, and uptake by macrophages in the intimal extracellular space, thereby promoting foam cell formation.

The observed higher affinity of LDL from survivors of myocardial infarction toward APG in in vitro binding assays^10–12 and our findings of a link between the presence of an atherogenic lipoprotein phenotype (ALP) and enhanced APG-LDL complex formation¹³ provided evidence that LDL in different individuals may be of variable atherogenicity. It is also increasingly accepted that VLDL and IDL have atherogenic potential. They have been isolated from atherosclerotic plaque and have been shown to enter the arterial wall and share with LDL the potential for causing lipid accumulation.^14–16 Their low efflux rate due to their large particle size¹⁵ augments the rate of cholesterol delivery to the arterial wall, and their impact can be significant, since they contain five times more cholesterol and cholesteryl ester per particle than LDL.¹⁷ The fact that they contain apoE as well as apoB may enhance their binding to APG.^18,19

The mechanism of interaction of APG with lipoprotein is not fully understood. It is believed that it could be a function of charge, sialic acid content,⁹ and/or the conformation of apoB.^13,19 Olsson et al⁸ found that a positively charged amino acid sequence on apoB that is rich in arginine and lysine was mainly responsible for this binding process. The same group of investigators have shown that neuraminidase treatment of LDL that removes sialic acid residues from apoB increases its binding reactivity with APG.⁹ In addition, we have recently demonstrated that the LDL of individuals with an increased percentage of LDLIII (small, dense LDL, which has been suggested to be more positively charged and have a lower sialic acid content than large LDL⁹) has a greater binding affinity toward APG¹³; an extension of the finding by Hurt-Camejo et al²⁰ that when APG was used to fractionate LDL, it had a preference for small, dense LDL. However, the determinants of reactivity variation in apoB-containing lipoproteins between individuals are still not clear, nor is it known what controls the relative reactivity of lipoprotein species throughout the S_f 0 to 400 spectrum.

Lipid-lowering treatment with fibrates affects both plasma triglyceride and cholesterol and is associated with a change in the LDL subfraction profile.²¹ It is predictable that such therapy affects lipoprotein-APG interaction, and it has recently been shown that drugs can reduce total LDL binding with APG.²² To explore further the mechanism of the binding process and to investigate further the concept that the atherogenecity of lipoprotein particles is linked to plasma triglyceride levels, we undertook a series of studies, the objectives of which were (1) to compare the reactivity of different apoB-containing lipoprotein subfractions with APG, (2) to examine the nature of this interaction and the effect of modifying lysine and arginine residues on LDL apoB interaction with APG, and (3) to test the effects of lipid-lowering treatment with ciprofibrate (which changes the LDL subfraction pattern) on different apoB-containing lipoprotein subfractions and their interaction with APG.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
1,2-Cyclohexandione, potassium cyanate, sodium borohydrate, formaldehyde, N-acetyl neuraminic acid (98% pure), and neuraminidase type VI-A from clostridium perfringens were purchased from Sigma-Aldrich Company Ltd (Poole, Dorset, UK). Phosphate-buffered saline tablets were from Unipath LTD (Basingstoke, UK). The membrane tubing used for the dialysis was Spectra/Por No. 2 (MW cutoff: 12 to 14 000) from Biologicals (Huntingdon, UK).

Subjects
A total of 32 subjects were recruited for all the studies. Each donated 50 mL of blood after an overnight (12-hour) fast. Blood was collected by venepuncture using K₂EDTA (final concentration 1 mg/mL) as an anticoagulant. Plasma was separated at 4°C by low-speed centrifugation (3000 rpm), and aliquots were used immediately for lipid, lipoprotein measurements, and LDL subfractionation.

Study I: The Reactivity of Different apoB-Containing Lipoproteins With APG
Eighteen subjects, aged 19 to 60 years, were divided into three groups (six subjects in each group) on the basis of their LDL subfraction profile and the presence or absence of ALP (Table 1). Group 1 (1 male and 5 females) were healthy, normolipidemic, aged 19 to 22 years, and had an LDL profile in which LDLI was the major species. Group 2 (4 males and 2 females) were healthy normolipidemic, aged 34 to 46 years, and had mainly LDLII. Group 3 were hyperlipidemic (5 males and 1 female), aged 39 to 60 years, with a profile in which LDLIII was the main subfraction.

View this table: [in this window] [in a new window]   Table 1. Lipid Variables and LDL Subfraction Distribution Within Total LDL for the Three Groups of Subjects

Study II: The Effect of Lipid-Lowering Treatment With Ciprofibrate on APG-Lipoprotein Interaction
Ten male patients, aged 40 to 60 years, with angiographically positive coronary artery disease (baseline total cholesterol >5.2 mmol/L, plasma triglyceride <3.5 mmol/L) were treated with ciprofibrate (100 mg/d for 8 weeks). Fasting plasma samples were taken before and after administration of the drug for 8 weeks.

The patients were not receiving any lipid-lowering medications at the time of recruitment and had no known or suspected hypersensitivity to the fibrate group of drugs. They had not suffered an MI in the previous 6 months; had no signs of renal, hepatic, or thyroid disease; were not diabetic; and consumed no more than 22 units of alcohol per week. Both studies were approved by the Research Ethics Committee of Glasgow Royal Infirmary, and each volunteer gave written informed consent.

Study III: LDL Modification and Its Effect on APG-LDL Complex Formation
Plasma from four subjects (mean total cholesterol 5.5 mmol/L, plasma triglyceride 1.6 mmol/L) was used to isolate by sequential density gradient centrifugation total LDL for chemical modification studies and neuraminidase treatment.

Lipoprotein Isolation and Plasma Lipid Measurement
In studies I and II, apoB-containing plasma lipoproteins (VLDL, IDL, and LDL) were subfractionated from fresh plasma by a modification of a previously published cumulative density gradient centrifugation procedure.²³ Six subfractions, two from each lipoprotein class, were obtained from each subject, ie, VLDL1 S_f (Svedberg flotation rate) (60 to 400), VLDL2 S_f (20 to 60), IDL1 S_f (16 to 20), IDL2 S_f (12 to 16), LDL A S_f (8 to 12), and LDL B S_f (0 to 8) (A and B were used to avoid confusion with LDLI, LDLII, and LDLIII). Centrifugation was performed over 48 hours at 23°C in a swinging bucket rotor (SW40; Beckman Industries, Inc.), and the rotor decelerated without braking. Lipoprotein subfractions were removed by a fine-tipped pipette from the top of the tube. The following centrifugation conditions were applied: 1 hour, 38 minutes at 39 000 rpm for VLDL1 (removed in a volume of 1 mL); 15 hr, 46 min at 18 500 rpm for VLDL2 (0.5 mL); 1 hour, 15 minutes at 39 000 rpm for IDL1 (0.5 mL); 1 hour, 22 minutes at 39 000 rpm for IDL2 (0.5 mL); 2 hours, 9 minutes at 39 000 rpm for LDLA (0.5 mL); and 17 hours at 40 000 rpm for LDLB (0.5 mL). The composition of free cholesterol, cholesteryl ester (determined as the difference between total and free cholesterol contents multiplied by 1.68 to allow for the fatty acid mass), triglyceride, and phospholipid of each lipoprotein fraction was measured by enzymatic methods as described,²⁴ and total protein was measured by a modification of the procedure of Lowry et al.²⁵ Lipoprotein concentration was calculated as the sum of these components (expressed as milligrams per deciliter of plasma). Apolipoprotein B content of each fraction was determined by isopropanol precipitation as described.²⁶ To divide the subjects into the three groups, their LDL subfractions were quantified in fresh plasma by nonequilibrium density gradient centrifugation using a six-step curvilinear salt gradient.²⁷ Three distinct LDL subfractions were resolved: large, LDLI (density 1.025 to 1.034 g/mL); intermediate, LDLII (density 1.034 to 1.044 g/mL); and small, dense, LDLIII (density 1.044 to 1.063 g/mL). Plasma cholesterol, triglyceride, VLDL, LDL, and HDL cholesterol were measured by using a modification of the Lipid Research Clinics Protocol.²⁸

LDL Modification
Total LDL (12 mL) was isolated from fresh plasma (24 mL) from four subjects (study III) by preparative sequential centrifugation at densities 1.019 to 1.063 g/mL in a fixed angle rotor (50.4 Ti; Beckman Industries, Inc.). Aliquots from this were taken for chemical and enzymatic modification and diluted in sodium borate buffer. Cyclohexandione modification of LDL arginine residues was achieved by the addition of 1,2-cyclohexandione and carbamylation of lysine residues by mixing with potassium cyanate as described previously.²⁹ Reductive methylation of lysine residues was performed by using sodium borohydride followed by the addition of 40% formaldehyde.²⁹ All modified samples were kept at 4°C overnight before dialysis and examination by agarose electrophoresis as described³⁰ to check for the extent of charge modification.

Neuraminidase Treatment of LDL
Neuraminidase treatment of LDL was performed by incubating total LDL, isolated by preparative sequential ultracentrifugation at densities 1.019 to 1.063 g/mL as above, with neuraminidase type VI-A from Clostridium perfringens for 25 hours at 37°C on a roller mixer as described.⁹ After incubation, the tubes were centrifuged for 1 minute at 1000 rpm, the supernatant was collected, and the pellet was washed with 0.5 mL of saline (0.15 mol/L NaCl). After recentrifugation, the supernatants were mixed and stored at 4°C overnight before dialysis for the APG binding assay and sialic acid measurement.

The effect of neuraminidase treatment on the integrity of apoB in LDL was tested on SDS-PAGE as described.³¹ Briefly, neuraminidase-treated and native LDL were mixed with 0.06 mol/L of Tris buffer, pH 8.5 (containing 10% glycerol, 1% SDS, and 5% ß-mercaptoethanol), heated for 15 minutes at 80°C, and applied to 0.75-mm-thick 4.0% to 22.5% polyacrylamide gel. After separation overnight at 30 mA and staining with 0.1 Coomassie blue,³¹ the proportion of B100 and other peptides was determined by scanning densitometry. The extent of charge modification of neuraminidase-treated LDL samples was assessed by agarose electrophoresis.³⁰

Isolation of Proteoglycans and APG-Lipoprotein-Binding Assay
APG was extracted by using guanidine-HCl from a single aorta from a 70-year-old deceased female less than 24 hours post mortem. This crude extract was further purified by CsCL₂ isopycnic density gradient centrifugation at P₀=1.5 g/mL.¹³ The bottom three fractions that had the highest chondroitin sulfate content as measured by alcian blue colorimetric assay³² and the highest LDL-complexing reactivity (data not shown) were pooled together.¹³ Binding of proteoglycan to LDL was studied by mixing varying amounts of CS-rich proteoglycans (0.5 to 5.0 µg chondroitin sulfate; all APG quantities are expressed on the basis of the chondroitin sulfate content) at pH 7.2 with 0.1 mg of LDL protein in a total volume of 1.1 mL. Details of the incubation procedure were described in a previous publication.¹³ Maximum LDL-APG-insoluble complex formation was achieved when APG containing 2.5 µg of CS was present in the incubation mixture. A standard solution of 25 µg/mL of CS-APG in 5.0 mmol/L of tris-HCl "binding" buffer, pH 7.2, containing 6.0 mmol/L of KCl, 4.0 mmol/L of CaCl_2, and 1.0 mmol/L of MgCl₂ was prepared, and 100 µL of this material was used in all binding assays.¹³

ApoB-containing lipoprotein fractions (VLDL1-LDLB) from the subject groups, before and after ciprofibrate treatment, modified LDL, and neuraminidase-treated LDL, were dialyzed at 4°C against the binding buffer described above.¹³ A 1.0-mL aliquot, diluted where necessary to contain 0.1 mg of protein from each sample, was mixed with 100 µL of CS-APG standard solution (2.5 µg of CS) and incubated at 25°C for 30 minutes. A separate aliquot was incubated without APG to serve as a control. The extent of insoluble complex formation was determined both as absorbance (due to turbidity) at 600 nm, the control tube having been subtracted as a blank, and as the percentage of precipitated cholesterol by measuring the cholesterol content of the APG-lipoprotein precipitate and dividing this by the total cholesterol content of the tube. Repeated analysis of the extent of insoluble complex formation using the same sample showed an intra-assay coefficient of variation of <3% and an interassay coefficient of variation of <6%. The content of precipitable apoB in a lipoprotein was calculated by determining for each subfraction the percentage of apoB precipitated in the assay (derived from the percentage of precipitated cholesterol multiplied by the apoB/cholesterol ratio) and multiplying this by the total apoB concentration of each subfraction in plasma.

Estimation of the amount of soluble complex formation between native or chemically modified LDL and APG was performed as described by Vijayagopal et al.⁴ Briefly, native or modified LDL (0.4 mg of protein) was mixed with 100 µg of CS-APG in a final volume of 2 mL at 25°C for 30 minutes. The solution density was then adjusted to 1.063 g/mL by adding D₂O and NaCl, and the mixture was subjected to centrifugation at 114 000g for 20 hours as described.⁴ After centrifugation, the tube content was fractionated into eight fractions. The cholesterol concentration of each fraction was determined by the enzymatic colorimetric CHOD-PAP method (Mannheim Boehringer kit 717/911),³³ and CS content was measured by the alcian blue colorimetric assay.³²

Sialic Acid Assay
Sialic acid determination was performed by using a modification of the resorcinol method of Svennerholm.³⁴ Briefly, a stock standard solution 32 µg/mL of N-acetyl neuraminic acid (Sigma) was prepared in distilled water. This was further diluted in distilled H₂O to give a working standard curve in the range 0 to 8 µg/250 µL. 250 µL of resorcinol reagent (0.2% resorcinol, 0.25 mmol/L CuSO₄, 8.2 mol/L HCl) was added to 250 µL of sample and N-acetyl neuraminic acid standard (0 to 8 µg/250 µL) in a glass screw-capped tube and incubated for 45 minutes at 100°C. After cooling in a water bath (5 minutes) at room temperature, 400 µL of butyl acetate:n-butanol (85:15 v/v) was added to each tube, vortexed, and centrifuged for 5 minutes at 3000 rpm. This was followed by absorbance measurement of the organic layer at 580 nm against a reagent blank.

Statistical Analysis
Statistical analysis and manipulation were performed by using MINITAB release 10 for Windows (Minitab, Inc, Pa). Pearsons correlation, univariate regression, two-sample t-test, and one-way ANOVA were used to assess the relationship between variables.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study I
Lipid and LDL Subfraction Profile
Mean plasma lipid level and LDL subfraction profile for the three groups of subjects are shown in Table 1. One-way ANOVA showed that plasma total cholesterol, triglyceride level, and LDL subfraction profile in group III subjects were significantly different from those in group I and group II subjects (P<.001). Group I and II had similar lipid levels but differed in their LDL subfraction profile P<.001 (Table 1).

The total mass of apoB-containing lipoprotein per 100 mL of plasma was significantly lower in groups I and II (327 mg/dL and 334 mg/dL, respectively) than in group III subjects (605 mg/dL) P<.001. Table 1 shows that there were no significant differences between the IDL1, IDL2, and LDLA concentration among the three groups of subjects.

ApoB-Containing Lipoprotein Subfractions and APG-Insoluble Complex Formation
In preliminary experiments, the linear range for reactivity (ie, insoluble complex formation) between apoB-containing lipoproteins and APG was determined by mixing varying amounts of lipoprotein fractions (VLDL1-LDLB, S_f 0 to 400) (0.025 to 0.2 mg of protein) with a fixed amount (2.5 µg) of CS-APG in a final volume of 1.1 mL. As seen in Fig 1A, the reactivity is linear up to a value of 0.1 mg of protein added to the assay. Above this, reactivity fell off; therefore, 0.1 mg of lipoprotein protein in 1 mL was used in the incubation. Up to a content of 0.1 mg of protein per assay, there was no difference in the slope of reactivity for the six apoB-containing fractions.

View larger version (18K): [in this window] [in a new window]   Figure 1. (A) Linearity of response in reactivity (AU 600 nm) to APG of lipoprotein subfractions. Varying amounts of lipoprotein fractions (VLDL1-LDLB, Sf 0 to 400) (0.025 to 0.2 mg protein) were mixed with a fixed amount (2.5 µg CS) of CS-rich APG (final volume of 1.1 mL) and incubated for 30 min at 25°C. The extent of lipoprotein-APG insoluble complex formation was determined by measuring turbidity at 600 nm. For each lipoprotein subfraction, the reactivity at 0.1 mg of protein added to the APG incubation was taken to be 100%. Reactivity of other dilutions of subfractions was expressed relative to this. (, VLDL1; , VLDL2; , IDL1; , IDL2; , LDLA; , LDLB.) (B) Correlation between turbidity as measured by absorbance unit (AU) at 600 nm and the amount of cholesterol precipitated measured after the dissociation of the pellet in 2.0 mol/L NaCl. % precipitated cholesterol is the amount of lipoprotein cholesterol precipitated by APG divided by the total cholesterol applied multiplied by 100.

The percentage of cholesterol in each lipoprotein fraction precipitated by APG correlated highly significantly (P<.000) with the turbidity measurement by absorbance at 600 nm (Fig 1B). Therefore, the latter was used in our analysis as the more convenient index for the amount of insoluble complex formed.

A similar pattern of lipoprotein-APG complex formation was observed in all individuals (Fig 2). The mean reactivity for the lipoprotein subfractions in the three groups was highest in IDL2 and LDLA, followed by LDLB and IDL1, and least reactivity was observed in the VLDL subfractions (Fig 2). The highest APG reactivity for a given amount of lipoprotein was found in group III, ie, those with raised lipid levels and a predominance of small, dense LDL. It was intermediate in group II, whose members were normolipidemic with LDLII as the main LDL species, and lowest in group I, which comprised young healthy subjects with a high LDLI level (Fig 2). When the amount of lipoprotein added to the incubation was expressed on the basis of apoB instead of the total protein, the pattern of reactivity was the same; insoluble complex formation as detected by turbidity at 600 nm was per 0.1 mg of VLDL1 apoB 0.18±0.01, per 0.1 mg VLDL2 apoB 0.14±0.01, per 0.1 mg IDL1 apoB 0.32±0.01, and 0.46±0.02, 0.45±0.01, and 0.30±0.04 for IDL2, LDLA, and LDLB apoB (0.1 mg each), respectively. The extent of APG-lipoprotein reactivity in each subfraction (except VLDL1) was positively related to plasma triglyceride level. Correlation coefficients between plasma triglyceride concentration and the extent of insoluble complex formation per 0.1 mg of protein for the six lipoprotein fractions were as follows: for VLDL1, r=.2 (NS); for VLDL2, r=.52 (P=.03); for IDL1, r=.82 (P<.0001); for IDL2, r=.68 (P<.01); for LDLA, r=.65 (P<.01); and for LDLB, r=.49 (P<.05).

View larger version (17K): [in this window] [in a new window]   Figure 2. Pattern of interaction of lipoprotein subfractions with arterial wall proteoglycans. The relative reactivity between the three different groups of subjects was compared by adding 0.1 mg protein to a fixed concentration (2.5 µg CS) of APG. The graph represents the median value for each lipoprotein subfractions with standard error bars in all the subjects, the line connects the median. (, group I; , group II; , group III. * P<.0001, ** P=.007.)

Study II
Effect of Ciprofibrate Treatment on APG-Lipoprotein Complex Formation
Lipid-lowering treatment with ciprofibrate resulted in a significant decrease in plasma cholesterol (18%, P=.039) and plasma triglyceride (44%, P<.05), a 10% reduction in LDL cholesterol, and a 9% increase in HDL cholesterol (Table 2). The changes in the last two variables did not reach statistical significance. There was a 46% increase in the percentage of LDLI and a 54% decrease in the percentage of LDLIII subfractions within total LDL after treatment with ciprofibrate (Table 2). No significant change was found in the LDLII subfraction. A significant reduction in the total plasma apoB-containing lipoprotein mass per 100 mL of plasma was found after lipid-lowering treatment (703±185 mg/dL compared to 524±140 mg/dL, P<.04).

View this table: [in this window] [in a new window]   Table 2. Mean±SD for the Patients' Lipid and LDL Subfraction Profile Before and After Treatment With Ciprofibrate

Treatment with ciprofibrate decreased the ability of each lipoprotein subfraction to form insoluble complexes with APG. For IDL1, IDL2, and LDLA, the reactivity per 0.1 mg of protein of lipoprotein was reduced significantly, by 54% (P=.03), 49% (P=.006), and 40% (P=.03) respectively, and that of LDLB was reduced by 42%, but this was not statistically significant (Fig 3). There was little or no change in the reactivity of the VLDL subfractions with APG (Fig 3). Since the drug changed both the quantity and the relative reactivity of the lipoprotein subfractions in circulation, total precipitability of the lipoprotein species was estimated before and on the drug as the product of the lipoprotein concentration (Table 2) in terms of its contained apoB and the proportion precipitated by APG (measured as the precipitated cholesterol). It was observed that the biggest changes were in the LDL (P<.001) range rather than in IDL (P<.05) (Fig 4), despite the fact that the relative reactivity was higher in IDL than LDL (Fig 3).

View larger version (15K): [in this window] [in a new window]   Figure 3. Lipoprotein-APG relative reactivity of the different lipoprotein subfractions before and after treatment with ciprofibrate (100 mg/d for 8 weeks) compared by adding 0.1 mg of protein to a fixed concentration (2.5 µg CS) of APG. The graph represents the median value for each lipoprotein subfractions with standard error bars in all the subjects; the line connects median values. (, before ciprofibrate; , after treatment with ciprofibrate. *P=.006, ** P=.03, *** P=.037.)

View larger version (15K): [in this window] [in a new window]   Figure 4. Total plasma apoB particles precipitated by APG in different lipoprotein fractions, before and after lipid-lowering treatment with ciprofibrate (100 mg/d for 8 weeks). 0.1 mg of protein from each lipoprotein fraction was added to a fixed amount of APG (2.5 µg CS), and the percentage of cholesterol precipitated as determined. This percentage was then multiplied by the total apoB content of the fraction off and on the drug. To compare the lipoprotein particle number more easily, the apoB precipitated was determined from the cholesterol to apoB ratio from compositional analysis of the fractions. (, before ciprofibrate treatment; , after treatment with ciprofibrate. *P<.01, **P<.05.)

Study III
LDL Modification and Its Effect on the Binding Reactivity with APG
Total LDL isolated from the plasma of normal volunteers by sequential gradient ultracentrifugation was subjected to a number of enzymatic and chemical modifications. Modification of lysine and arginine residues by carbamylation and cyclohexandione treatment, respectively,²⁹ reduced the net positive charge of LDL on agarose electrophoresis (data not shown) and abolished the ability of the LDL to form complexes with APG. Reductive methylation, which did not alter the charge on LDL, was also effective in completely blocking the binding reactivity of LDL with APG (reactivity of native LDL was 0.576±0.015, while that of reductively methylated LDL was 0.006±0.001).

It was possible that modification of LDL led to the formation of soluble complexes that were not detected in the turbidity assay.⁴ To exclude this, chemically modified and native LDL (0.4 mg of protein) from four different subjects were incubated with 100 µg of CS-APG in the binding buffer, pH 7.2, for 30 min at 25°C. After centrifugation at density 1.063 g/mL for 20 hours, eight fractions were collected from bottom to top of the tube, and each fraction was analyzed for CS by alcian blue colorimetric assay³² and for cholesterol.³³ When cyclohexandione-treated, carbamylated, or reductively methylated LDL was incubated with CS-APG, the bottom fraction after centrifugation contained 98% to 100% of the starting CS-APG, and the top fraction contained 100% of the starting cholesterol. In comparison, when native LDL from the same subjects was used, 60% of the CS-APG was in the bottom fraction, while the top fraction contained 100% of the cholesterol and 35% of the CS-APG. In keeping with the findings reported by Vijayagopal et al,⁴ this indicated that no soluble complexes were formed when any of the chemically modified LDL preparations was incubated with APG.

Sialic Acid Content and APG Binding
The content of sialic acid per apoB varied significantly between the different lipoprotein subfractions, being highest in VLDL1 and lowest in LDL-B in all subjects (Fig 5). The two LDL subfractions in each individual had similar sialic acid content (19.7±5.2 µg and 19.4±5.4 µg per particle for LDLA and LDLB, respectively). The sialic acid content of LDLA and LDLB was significantly higher in group I subjects than in group III subjects and was inversely related across all subjects to the extent of LDL-insoluble complex formation with APG (data not shown). When the sialic acid content in the other lipoprotein subfractions was examined, no significant differences were found among the three groups.

View larger version (16K): [in this window] [in a new window]   Figure 5. The amount of sialic acid content of the different lipoprotein subfractions among the three different groups of subjects. (, Group I; , group II; , group III.)

Incubation of LDL with neuraminidase for 25 hours released up to 50% of the sialic acid present on LDL. This resulted in a 54% increase in the binding reactivity of the LDL for CS-APG compared to the control sample. To eliminate the possibility that protease contamination of the neuraminidase influenced the results, the apoB component of neuraminidase treated LDL was examined on SDS-PAGE. No evidence was found for degradation after exposure of LDL to the enzyme.

A significant reduction in the sialic acid content of all lipoprotein subfractions was found after treatment with ciprofibrate for 8 weeks (data not shown). However, this change in sialic acid content was not correlated to the change in lipoprotein-APG interaction (r=.01, NS).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Structural diversity exists throughout the apoB-containing lipoprotein spectrum. High-resolution centrifugation or electrophoretic techniques have been used to demonstrate the presence of discrete subfractions within VLDL,^23,35 IDL,³⁶ and LDL.^27,37 These fractions all have varying functional and metabolic properties and are likely to differ in their atherogenic potential. LDL, in particular, has been shown to consist of discrete subpopulations of particles, of which the small, dense species have been most closely linked to increased risk of CHD.³⁷ The observation that the strength of the interaction between LDL and APG was related to the subfraction profile¹³ and the knowledge that small, dense LDL was but one component of the dyslipidemia termed the atherogenic lipoprotein phenotype³⁸ prompted us to seek further abnormalities within the S_f 0 to 400 lipoprotein spectrum. To use a single uniform isolation procedure for all apoB-containing lipoproteins, LDL was subfractionated by cumulative centrifugation into LDLA (S_f 8 to 12; d=1.020 to 1.038), which comprised LDLI plus a portion of LDLII and LDLB S_f (0 to 8) (d=1.039 to 1.060 g/mL,) which contained the remainder of LDLII plus LDLIII.

The principal finding of the present study was that in an individual small, dense IDL and large LDL were the most reactive species toward APG. This contradicted our initial supposition that there would be a monotonous increase in APG reactivity from VLDL1 through to LDLB. If APG trapping of lipoprotein is, as we believe, a key early step in atherogenesis, then the data provide strong support for the suggestion that IDL is a particularly atherogenic lipoprotein.^39–43 The general pattern of APG-lipoprotein interaction was remarkably similar in all subjects, regardless of their plasma lipid levels (Fig 2). However, between individuals, the relative reactivity of all lipoprotein subclasses increased as plasma triglyceride and the proportion of LDLIII rose. Furthermore, in subjects treated with ciprofibrate, the reactivity of all species fell in concert, with again the same general pattern being maintained. The basis of lipoprotein-APG interaction throughout the S_f 0 to 400 spectrum is unknown, although for LDL the work of Olsson et al⁸ pinpointed certain sequences of apoB as important. What the data in Figs 2 and 3 suggest is that the relative reactivities of VLDL2, IDL1, IDL2, LDLA, and LDLB are linked, probably by a common denominator.

The findings also provide further insight into possible association between plasma triglyceride concentration and CHD risk. On the basis of current knowledge, two mechanisms can be postulated to explain the link between the plasma triglyceride concentration and lipoprotein-APG reactivity. First, high levels of plasma triglyceride may cause remodeling of IDL and LDL to more atherogenic forms. As plasma triglyceride rises, so does the extent of cholesterol ester transfer protein–mediated triglyceride exchange into denser lipoproteins such as LDL and HDL. Lipolysis of these triglyceride-enriched particles results in the generation of smaller and denser lipoproteins.^44,45 It is tempting to speculate that IDL is affected in the same way and that small, dense IDL are active in binding APG. Second, it can be argued that APG reactive species within the IDL and LDL density intervals are the products of the lipolysis of large VLDL1. Metabolic studies have shown that VLDL1 is converted to VLDL2 remnants and IDL and LDL particles that have a prolonged residence time in circulation compared to lipoproteins whose initial precursor is in the VLDL2 density range.³⁶ The properties of IDL and LDL particles that circulate for a long time in plasma may be modified, for example by altered surface glycosylation or oxidation, to enhance their APG binding. When VLDL1 levels fall on ciprofibrate (the major drug-induced change in the S_f 0 to 400 lipoprotein), possibly owing to decreased hepatic secretion,⁴⁶ so does the relative reactivity of IDL and LDL.

How lipoproteins bind to and are precipitated by APG is not clear. Extensive studies by Camejo et al⁹ linked APG reactivity to the overall charge on the lipoprotein; ie, binding is basically an electrostatic phenomenon. This explains the effects noted here and earlier⁹ of modifying the sialic acid content of LDL and blocking arginine and lysine residues using cyclohexandione and cyanate, respectively. However, the sialic acid content of the different lipoprotein subfractions within an individual did not significantly correlate with the APG-lipoprotein complex formation (in keeping also with the finding that Lp(a) contains more sialic acid but has a higher reactivity toward APG than toward LDL⁵). In addition, the observation that reductive methylation (as for LDL receptor binding) blocks interaction with APG indicates that the nature of the reaction may be more subtle than simple electrostatic interaction. This modification does not alter the charge on the lipoprotein but does prevent lysine groups entering into hydrogen bonding and possibly induces conformational change in apoB. It is worth noting that reductive methylation was reported by Mahley et al¹⁸ not to affect heparin (analogous to heparan sulfate) binding of LDL and HDL. Further, marked differences in the ability of heparin and chondroitin sulfate proteoglycan to form soluble and insoluble complexes with LDL have been reported.⁴⁷ It is tempting to speculate that clusters of positively charged residues on apoB must be in the correct conformation to facilitate APG binding and that this conformation is favored in the apoB-containing lipoproteins of subjects with small, dense LDL. We conclude, therefore, that an ALP leads to abnormalities throughout the apoB-containing lipoprotein spectrum and that the enrichment of VLDL2, IDL, and LDL in lipoprotein species that bind avidly to APG is one of the ways in which elevated plasma triglyceride levels contribute to the atherogenic process. These abnormalities are corrected by appropriate lipid-lowering therapy.

	Acknowledgments

 
We thank the staff of the routine lipid section of the Department of Pathological Biochemistry, Glasgow Royal Infirmary, for plasma lipid analysis. Our gratitude is also due to Dr Ian Hutton at the Department of Cardiology, GRI, for allowing to recruit hyperlipidemic subjects directly under his care. Ciprofibrate was a generous gift from Sanofi Winthrop Ltd. V. Anber is supported by Scotia pharmaceuticals, and J. Millar is supported by a grant (G9307710PA) from the MRC.

Received April 7, 1997; accepted August 18, 1997.

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