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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3021-3032

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3021-3032.)
© 1997 American Heart Association, Inc.

Articles

Apolipoprotein A-IFIN (Leu159->Arg) Mutation Affects Lecithin

Cholesterol Acyltransferase Activation and Subclass Distribution of HDL but Not Cholesterol Efflux From Fibroblasts

Helena E. Miettinen; Matti Jauhiainen; Helena Gylling; Sonja Ehnholm; Ari Palomäki; Tatu A. Miettinen; ; Kimmo Kontula

From the Department of Medicine, University of Helsinki, Helsinki, Finland (H.E.M., H.G., T.A.M, K.K); the Department of Biochemistry, National Public Health Institute, Helsinki, Finland (M.J., S.E.); and the Department of Cardiology, Central Hospital of Hämeenlinna, Finland (A.P.).

Correspondence to Helena E. Miettinen, MD, Department of Medicine, University of Helsinki, Meilahti Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland. E-mail Helena.Miettinen{at}helsinki.fi


*    Abstract
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Abstract We showed earlier that the apolipoprotein A-I Leu159->Arg mutation (apoA-IFin) results in dominantly inherited hypoalphalipoproteinemia. In the present study we investigated the effect of the apoA-IFin mutation on lipoprotein profile, apoA-I kinetics, lecithin:cholesterol acyltransferase (LCAT) activation, and cholesterol efflux in vitro. Carriers (n=9) of the apoA-IFin mutation exhibited several lipoprotein abnormalities. The serum HDL cholesterol level was diminished to 20% of normal, and nondenaturing gradient gel electrophoresis of HDL showed disappearance of particles at the 9.0- to 12-nm size range (HDL2-type) and the presence of small 7.8- to 8.9-nm (mostly HDL3-type) particles only. HDL3-type particles from both the mutation carriers and nonaffected family members were similarly converted to large, HDL2-type particles by phospholipid transfer protein in vitro. Studies on apoA-I kinetics in four affected subjects favored accelerated catabolism of apoA-I. Experiments with reconstituted proteoliposomes showed that the capacity of apoA-IFin protein to activate LCAT was reduced to 40% of that of the wild-type apoA-I. The impact of the apoA-IFin protein on cholesterol efflux was examined in vitro using [3H]cholesterol-loaded human fibroblasts and three different cholesterol acceptors: (1) total HDL, (2) total apoA-I combined with phospholipid, and (3) apoA-I isoform (apoA-IFin or wild-type apoA-I isoform 1) combined with phospholipid. ApoA-IFin did not impair phospholipid binding or cholesterol efflux from fibroblasts to any of the acceptors used. Only one of the nine apoA-IFin carriers appears to have evidence of clinically manifested atherosclerosis. In conclusion, although the apoA-IFin mutation does not alter the properties of apoA-I involved in promotion of cholesterol efflux, its ability to activate LCAT in vitro is defective. In vivo, apoA-IFin was found to be associated with several lipoprotein composition rearrangements and increased catabolism of apoA-I.


Key Words: reverse cholesterol transport • HDL • apolipoprotein A-I kinetics • coronary artery disease • mutation


*    Introduction
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 *Introduction
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The inverse relationship between serum HDL-C concentration and risk for CAD is well established.1 The power of low HDL-C, especially of its subfraction HDL2,2 as a predictor of CAD has been enlightened by several epidemiologic studies and clinical trials.3 However, the exact mechanisms underlying these associations have remained unsolved. It is still a matter of debate whether HDL itself possesses a direct role in prevention of atherosclerosis. HDL and apoA-I, the major protein component of HDL, are able to promote cholesterol efflux from cells in vitro4–6 and thus hold an important position in RCT,7,8 a process now widely accepted as antiatherogenic. ApoA-I is also the most potent activator of LCAT, an enzyme responsible for esterification of cell and lipoprotein-derived cholesterol; this enzymatic reaction appears to be a crucial step in RCT, creating a concentration gradient and flux of free cholesterol into HDL.7 Evidence for the cardioprotective nature of apoA-I has further been provided by animal studies: transgenic mice overexpressing human apoA-I have elevated HDL-C levels and are protected against diet-induced fatty streak formation.9 On the other hand, apoA-I knockout mice, lacking apoA-I in plasma and having drastically reduced HDL-C concentrations, were not found to be more prone to the development of atherosclerosis than wild-type mice.10

Human genetic HDL deficiency syndromes11 associated with extremely low serum HDL-C levels provide a unique opportunity to further investigate the in vivo role of HDL and apoA-I in protection against premature CAD. Genetic factors influencing structural or functional properties of apoA-I, LCAT, and thereby HDL could have an impact on RCT and thus influence the liability for CAD. However, although there are several inherited genetic disorders in which HDL-C and apoA-I levels are severely reduced, it is very seldom that they seem to be associated with increased risk of premature atherosclerosis.12

Although several mutations in the apoA-I gene have been identified, 12 only a few of them cause extreme forms of hypoalphalipoproteinemia.13–24 Typically the phenotype is inherited in a codominant fashion: affected subjects are homozygous for the mutation, and heterozygous carriers have normal or approximately half normal HDL-C and apoA-I concentrations. However, there are a few reports on specific apoA-I gene mutations that have a dominant impact on the phenotypic expression of HDL-C levels.16,17,24–26 ApoA-IMilano, the first apolipoprotein variant ever discovered, substitutes cysteine for arginine at the residue 173 of apoA-I, thus providing the mutant protein with the capability to form dimers. Carriers of the mutation have reduced serum HDL-C and apoA-I levels (33% and 60% of normal, respectively) and slight hypertriglyceridemia, but they do not seem to be at risk for premature CAD.27 ApoA-ISeattle, a de novo mutation detected in one individual only, deletes amino acids 145 to 160 of the apoA-I protein and diminishes HDL-C and apoA-I concentration below 15% of the normal.17 We have previously described the apoA-IFin (Leu 159->Arg) mutation causing dominantly inherited hypoalphalipoproteinemia in a large kindred from Finland.16 ApoA-IFin exerts an even more profound effect on serum HDL-C and apoA-I levels than A-IMilano, and heterozygous carriers of apoA-IFin have HDL-C and apoA-I levels reduced to 20% and 25% of that of the nonaffected family members.

The present study was undertaken to further enlighten the structure-function relationships of apoA-I by examining the effect of apoA-IFin on overall lipoprotein profile, HDL particle size and composition, apoA-I kinetics, and LCAT activation. To gain more insight into the possible atherogenicity/antiatherogenicity of apoA-IFin, we extended our previous pedigree analysis16 and examined the impact of apoA-IFin on cholesterol efflux in vitro.


*    Methods
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Patients
The study group consisted of a large kindred with severe hypoalphalipoproteinemia (Fig 1Down) described before,16 now further extended by three new family members (members 16 to 18). All affected subjects had similarly reduced HDL-C, apoA-I, and apoA-II concentrations (approximately 20%, 25%, and 50% of those of the nonaffected members, respectively) and were carriers of the apoA-IFin mutation. Clinical histories were recorded, and physical examinations were performed for each of the family members.



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  		Figure 1. Pedigree of the family.

Measurement of Serum Lipids and Lipoproteins
Total cholesterol, free cholesterol, triglycerides, and phospholipids were determined enzymatically using commercial kits (Hoffmann-La Roche, Boehringer Mannheim, and Wako Chemicals GmbH). Apolipoproteins apoA-I, apoA-II, and apoB were measured with immunochemical assays (Orion Diagnostica). Serum lipoproteins were separated by ultracentrifugation into density classes.28 HDL (from all family members) and LDL fractions (from four affected and two nonaffected members) were further divided into subfractions according to their densities by ultracentrifugation; HDL into HDL2 and HDL3 fractions (d=1.063 to 1.125 g/mL and d=1.125 to 1.21 g/mL, respectively) and LDL into light, dense, and ultra-dense LDL fractions (d=1.019 to 1.036, 1.037 to 1.055, and 1.056 to 1.063 g/mL, respectively).29 The separated lipoprotein fractions were not corrected for recovery.

ApoA-IFin Genotyping
Genotyping of the new family members was performed by PCR amplification of exon 4 of the apoA-I gene, followed by digestion of the amplification products with the restriction enzyme Fsp I as previously described.16 In short, PCR was performed using primers 5'-AAGGTGCAGCCCTACCTG-3' and 5'-TTTATTCTGAGCACCGGGAA-3' and 30 amplification cycles at 95, 58, and 72°C for 1 minute each. The PCR product, 487 bp in size, was digested with the restriction enzyme Fsp I (New England Biolabs) and run on a 2.5% agarose gel. The T-to-G base substitution destroys an existing Fsp I cutting site, resulting in an amplification product 487 bp in size, while the normal allele produces two fragments (294 and 193 bp) on digestion with Fsp I.

HDL Particle Analysis
Fresh HDL fractions (5 µg of protein/well), separated by ultracentrifugation from 7 affected and 16 unaffected subjects, were run on self-casted nondenaturing polyacrylamide gradient gels (4% to 22%) using a Mini-Protean II Cell electrophoresis apparatus (Bio-Rad). Gels were casted using a model 385 gradient former (Pharmacia) and a model EP-1 Econo Pump (Pharmacia). A control HDL sample (stored in aliquots at -70°C) was included in each electrophoresis. Commercial protein standards (Pharmacia) were run in three lanes of the gels (in the center and in both sides) to ensure calibration of particle sizes. The electrophoresis running buffer (pH 8.4) consisted of 90 mmol/L Tris base, 80 mmol/L boric acid, 3 mmol/L EDTA and 3 mmol/L NaN3. Before loading, HDL samples were diluted with loading buffer containing 40% sucrose and 0.05% bromphenol blue. Electrophoresis of the samples was carried out at +4°C for 10 minutes with 40 V and continued for 4 hours with a constant voltage of 200 V. Proteins were then detected with Coomassie brilliant blue G-250 (Bio-Rad) and destained with 5% acetic acid. Gels were scanned and analyzed with the Bio Image system (Millipore Co), and particle sizes were determined using commercial protein standards (Pharmacia).

Measurement of CETP and PLTP Activity and HDL Conversion Experiments
CETP activity was measured according to Groener et al30 from two affected and two nonaffected family members. Individual values were calculated as the CETP activity measured from the sample divided by the activity of the internal control and expressed as arbitrary units. PLTP was purified and assayed as described previously.31 Purified PLTP preparations were free of CETP, LCAT, and hepatic lipase and gave a single band of 78 to 80 kDa when analyzed by SDS-PAGE. Proteoliposomes for PLTP assays were prepared using 10 µmol of egg PC, 1 µCi of [14C]dipalmitoyl phosphatidylcholine, and 20 nmol of butylated hydroxytoluene. PLTP assays were carried out in a volume of 400 µL containing HDL3 (250 µg of protein), 150 nmol of [14C]PC-labeled liposomes, plasma sample (4 µL of plasma/incubation, plasma first diluted 1:10 in TBS) and TBS, pH 7.4.

The capability of PLTP to convert HDL3-type particles to large and small particles was examined by incubating native HDL3 or total HDLFin (250 µg of protein/incubation) in the absence and presence of PLTP (500 nmol/h/incubation, corresponding to approximately 0.6 µg of total protein) for 24 hours at +37°C as previously described.31 Incubations were carried out in a volume of 0.4 mL using a buffer containing 10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl and 1 mmol/L EDTA (TBS). After incubations, the tubes were placed on ice, and the particle sizes were analyzed as described above.

Kinetic Studies
Kinetic studies were performed as previously described.32 In brief, autologous apoA-I was isolated from total HDL with guanidine hydrochloride incubation and iodinated with 125I by a modification of the iodine monochloride method.33 The total amount of radioactivity per injected 125I-apoA-I did not exceed 20 µCi. After the injection, blood samples were collected and counted for 14 days. The die-away curves for the tracer were constructed, and FCR was determined from these data using a two-pool model.34 Transport rate was determined by multiplying FCR by the respective pool size (calculated using serum apoA-I values) of the tracer. Plasma volume was calculated as 4.5% of body weight.

Isolation of ApoA-IFin
Plasma of a heterozygous carrier of the apoA-IFin mutation was used as a source to isolate the apoA-IFin protein. HDL was isolated from plasma (400 mL, obtained by plasmapheresis) of both an affected family member and a normal control subject by a series of ultracentrifugations.28 ApoA-I was purified from HDL using guanidine hydrochloride as described previously,35 dialyzed against 5 mmol/L NH4HCO3, and lyophilized. Lyophilized apoA-I samples (10 mg) were dissolved in 1 mL of a buffer containing 0.01 mol/L Tris-HCl (pH 7.4) and 1% decylsulfate. Separation of the apoA-IFin from its normal apoA-I counterpart and control apoA-I from its normally occurring second isoform was carried out by preparative IEF. Preparative IEF gels were made as previously described36 with the separating gradient covering the pH range 4 to 6.5. After overnight electrophoresis, the gels were covered with H2O, and the separated protein bands were visualized under white light. Protein bands were cut from the gels, followed by elution of proteins from gel slices with 4 mol/L guanidine hydrochloride. Protein preparations were dialyzed against 5 mmol/L NH4HCO3 for 16 hours, lyophilized, and resolubilized into phosphate-buffered saline. Protein concentrations were determined as described above, and aliquots were stored at -20°C until use. Analytical IEF was used to confirm the authenticity of the appropriate isoforms.

LCAT Activity Assay
LCAT was isolated from fresh human plasma using a method described earlier37 with the following modifications38,39: the anion exchanger Mono Q HR 5/5 (Pharmacia) was used instead of DEAE-Sephadex A-50, and the final purification step was hydroxyl apatite chromatography on Bio-Gel HTP (Bio Rad). The purified LCAT preparation exhibited a single band on SDS-PAGE with an apparent molecular mass of 68 kDa and was free of CETP, PLTP, and hepatic lipase. LCAT preparations were stored at +4°C in TBS buffer until use.

ApoA-I-egg PC-[1,2-3H]cholesterol proteoliposomes were prepared by the cholate dialysis method as described.40 Four different apoA-I preparations were used: Control apoA-I (a gift from Swiss Red Cross, Central Laboratory, Switzerland), total apoA-I from an apoA-IFin carrier, wild-type apoA-I isoform isolated by IEF, or apoA-IFin isoform isolated by IEF. Egg PC and [3H]cholesterol in organic solvent were mixed, and organic solvents were evaporated under nitrogen followed by 30 minutes of lyophilization. The dried lipids were resolubilized by vortexing in TBS. ApoA-I and sodium cholate (final concentration 55 mmol/L) were then added, and the mixture was vortexed for 1 minute and incubated at +25°C for 30 minutes. Sodium cholate was then removed by dialyzing against TBS for 72 hours. Molar ratios of proteoliposome components were determined by measuring cholesterol and phospholipids by enzymatic methods and apoA-I by immunoturbidometry. Analysis of substrate compositions showed that all four proteoliposome preparations consisted of equal molar ratios of apoA-I, PC, and cholesterol (0.74±0.016, 262±6.74, and 8.9±0.29, mean±SE, respectively). All proteoliposomes were of similar size when evaluated with gel filtration in a Superose 6HR column, and the recoveries from the column were about 90%. Proteoliposomes were stored at +4°C under nitrogen and were used within 2 weeks.

LCAT activity was measured as described earlier38 using the freshly prepared proteoliposomes. In brief, the assay mixture consisted of 250 µL of assay buffer (TBS), 125 µL of 2% bovine serum albumin and 50 µL of egg PC-cholesterol-apoA-I proteoliposome substrate (corresponding to 11 µg of apoA-I/assay). The mixture was preincubated for 20 minutes at +37°C, after which 25 µL of 0.1 mol/L 2-mercaptoethanol and 25 to 50 µL of purified LCAT enzyme were added. The mixture was vortexed and incubated at +37°C for 30 minutes, and the reaction was stopped by adding 8 mL of chloroform-methanol (2:1, v/v). Lipids were extracted, the organic phase of the extract was dried under nitrogen and dissolved in chloroform, and the lipids were separated by thin-layer chromatography. Radioactivities in the cholesterol and cholesterol ester regions were quantitated using a liquid scintillation counter. Comparison was made to a control apoA-I preparation with LCAT activity of 60±2 nmol/h/mL, which was set as 100%. The initial rates of the enzymatic reaction with purified LCAT were determined by using an enzyme dilution that resulted in a linear reaction rate.

Reconstituting HDL Discs for Efflux Experiments
Reconstituted HDL discs used in efflux experiments were prepared by the cholate dialysis method using DMPC as described previously.40,41 Molar ratios of apoA-I (either total apoA-I from an affected and nonrelated nonaffected subjects or only the mutant or wild-type isoforms isolated by IEF), DMPC, and cholate were 1:150:300. ApoA-I, DMPC, and cholate mixtures were incubated for 12 hours at +37°C, followed by removal of cholate by dialysis against TBS for 72 hours. Reconstituted particles were analyzed on native polyacrylamide gradient gels (as described above) to confirm that apoA-IFin binds phospholipids and is able to form discoidal particles. Discs were stored at +4°C and used within 2 weeks.

Cholesterol Efflux In Vitro
Human embryonic fibroblasts (HES cells) were grown in Dulbecco's modified Eagle's medium containing 4 mmol/L glutamine, streptomycin, penicillin, and 10% fetal calf serum in 35-mm cell culture plates. Cells were grown until subconfluent and starved overnight in serum-free OPTI-MEM (Gibco BRL).3H-labeled cholesterol was incubated with 20% human albumin at + 37°C and vortexed briefly, and the resulting [3H]cholesterol-albumin mixture (600 000 cpm/plate) was then added onto washed cells. Cells were labeled overnight in serum-free media (OPTI-MEM), washed three times with PBS, and incubated for 1 to 24 hours in serum-free OPTI-MEM with various reconstituted HDL discs (2.5 to 150 µg/mL) or native HDL (150 to 450 µg/mL). Media were collected, and cells were washed and lysed in 1 mL of 0.5 mol/L NaOH. After determination of radioactivities by liquid scintillation counting, cholesterol efflux was expressed as the percentage of radioactivity present in the medium in relation to the initial cellular radioactivity. The cholesterol efflux capacity of the medium alone (accounting for 10 to 15% of the efflux activity of the acceptors under study) was determined in each assay and subtracted from the data before their analysis.

Statistical Methods
Mean levels of serum lipids and lipoproteins were compared using Student's t test. In the case of serum and lipoprotein triglyceride levels, log transformations of the lipid data were carried out before comparison.


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Clinical Data and Lipoproteins
Clinical examination did not reveal corneal clouding, lipid arcus, or skin or tendon xanthomas in any of the affected subjects. None of the previously described family members (1 to 15), nor additional members 17 and 18, had clinical evidence of CAD (exercise-induced angina, past history of myocardial infarction, ECG abnormalities suggestive of ischemia, or a history of coronary bypass operation). However, affected family member 16 had undergone a coronary bypass operation at the age of 60. He had no other known risk factors for CAD, except low serum HDL-C due to the apoA-IFin mutation. His son, family member 17 and carrier of the mutation, was without clinical signs of CAD, and his exertion ECG was normal.

Serum lipid and lipoprotein profiles (Tables 1Down and 2Down) showed comparable levels of serum total cholesterol and phospholipids in affected and nonaffected family members but slight hypertriglyceridemia in affected ones. The amounts of triglycerides and phospholipids present in the LDL fraction were higher in the affected subjects (75 and 110 mg/dL, respectively) than in the nonaffected subjects (20 and 70 mg/dL, respectively). LDLs of four affected and two nonaffected subjects were further divided into three fractions according to their densities (light, dense, and ultra-dense); all LDL fractions of affected family members showed similar increments of triglycerides as well as low cholesterol:triglyceride ratios (data not shown). However, mean±SE LDL apoB concentrations were similar in affected (68.0±8.0 mg/dL) and nonaffected individuals (69.2±1.0 mg/dL); neither were there any significant differences in apoB concentrations of the LDL subfractions in these two groups (data not shown). These data indicate that the principal abnormality of LDL is its enrichment with triglycerides. IDL fractions of the affected subjects likewise had a high triglyceride content (Table 1Down).


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  		Table 1. Serum and Lipoprotein Total Cholesterol and Triglyceride Levels in Affected and Nonaffected Family Members


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  		Table 2. Serum and Lipoprotein Phospholipid and Apolipoprotein Levels in Affected and Nonaffected Family Members

HDL particle analysis of the affected family members showed not only quantitative (Tables 1Up and 2Up) but also compositional changes in all HDL fractions (Table 3Down). The concentration of total HDL-C was reduced to 20%, apoA-I to 25%, and apoA-II to 50% of that of the nonaffected subjects. Although HDL phospholipids and triglycerides were reduced in mutation carriers, their HDL was, however, disproportionally rich in phospholipids and triglycerides in relation to the amount of cholesterol present (Table 3Down). Reduction of HDL-C was particularly significant in the HDL2 density range, with most of the HDL-C being present in the HDL3 density range in affected subjects (Table 1Up). Because the serum apoA-II concentration in the mutation carriers was diminished to a lesser extent than that of apoA-I, the ratio of apoA-II to apoA-I in HDL and its subfractions was high. ApoA-IFin carriers also showed high ratios of phospholipid to apoA-I in their HDL. Cholesterol ester percentage in serum was lower in the nine affected (68.3±1.4%) than in the nine nonaffected family members (74.8±0.8%, P<.01). There was a similar trend in cholesterol ester percentages in HDL (76.0±3.7 versus 83.2±0.7%, P=.07) whereas the cholesterol ester percentages in LDL were similar in the two groups.


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  		Table 3. HDL Particle Compositions in Affected (n=9) and Nonaffected (n=9) Family Members

Analysis of HDL Particle Size
To examine the HDL particle sizes in more detail, HDL samples from 16 nonaffected and 7 affected subjects were electrophoresed on native polyacrylamide gradient gels (4% to 22%) and stained with Coomassie brilliant blue. The data from 7 affected and 7 nonaffected family members are summarized in Table 4Down. Computer analysis of the scanned gels revealed that the most prominent characteristic in the affected subjects was an almost total lack of HDLs at the size range 8.9 to 12.0 nm, corresponding to HDL2a,b particle diameter range. HDLs from the affected subjects were detected only within the size range of 7.8 to 8.9 nm, consistent with the HDL3 subfraction range. The mean±SE HDL particle size in the affected subjects was 8.1±0.09 nm. The 16 nonaffected family members had both HDL2-size particles (10.7±0.14 nm; range, 9 to 11.35 nm) and HDL3-size particles (8.0±0.06 nm; range, 7.6 to 8.8 nm).


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  		Table 4. HDL Particles from Seven Nonaffected and Seven Affected Family Members Estimated From the Scanned and Computer-Analyzed Nondenaturing Polyacrylamide Gradient Gels

PLTP-Mediated HDL Conversion and CETP Activation
Absence of HDL2-size particles in mutation carriers may have resulted from impaired conversion of HDL3 to HDL2 by PLTP. This was investigated by incubating HDL from an affected and a nonaffected subject with purified PLTP and subsequent analysis of the resulting particles on native polyacrylamide gradient gel. Analysis of the scanned gel by computer (Bio Image system) (Fig 2Down) showed a similar appearance of larger, HDL2-size particles in both the affected and the nonaffected subjects. Moreover, subsequent formation of small particles was seen in both the affected and nonaffected subjects (Fig 2Down). These particles were shown to have pre-ß mobility in agarose gel (data not shown).



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  		Figure 2. Nondenaturing gradient gel electrophoresis of total HDL from an A-IFin carrier and HDL3 from a control subject after incubation with and without PLTP. Lane 1, protein standards with diameters (from top to bottom) 17, 12.2, 10.4, 8.1, and 7.1 nm; lanes 2 and 3, HDLFin with and without PLTP, respectively; lanes 4 and 5, control HDL3 with and without PLTP, respectively.

PLTP activity in two affected subjects (4200 and 4100 nmol PC transferred/mL/h) was normal in comparison to the activity in the normolipidemic control subjects (3850±490 nmol/mL/h). However, CETP activity, measured from two affected and two nonaffected family members, was found to be reduced by approximately 50% in the affected subjects (0.418 and 0.441 versus 0.857 and 0.966 arbitrary units).

Kinetic Studies of ApoA-I
To further clarify the mechanism underlying low apoA-I levels, the kinetics of apoA-I was investigated in 2 normotriglyceridemic affected family members. Data of the previous and present kinetic studies are summarized in Table 5Down. Altogether, apoA-I kinetic studies were performed with 4 affected family members and a control group comprising 13 unrelated subjects. Affected family members had higher apoA-I FCRs in comparison with the upper 95% confidence limit of the control subjects. In addition, transport rate of apoA-I was subnormal in 3 of the 4 affected subjects.


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  		Table 5. ApoA-I Kinetics in Four Affected Subjects and 13 Unrelated Control Subjects

LCAT Activation In Vitro
LCAT activation experiments were carried out with reconstituted proteoliposomes as substrates. Activation of LCAT was determined using the following apoA-Is: total apoA-I from an apoA-IFin mutation carrier, purified apoA-IFin (isolated and purified by IEF) and purified normal apoA-I isoform 1 from a nonaffected subject (isolated and purified by IEF), as well as a highly purified control apoA-I. The possibility that apoA-IFin could have influenced particle formation by altering protein-phospholipid interaction was excluded by examining proteoliposomes by gel filtration; all proteoliposome particles were of the same size. LCAT activation experiments using apoA-IFin proteoliposomes revealed a significant reduction in LCAT activation capacity in comparison with proteoliposomes containing either wild-type apoA-I isoform or total apoA-I from a heterozygous apoA-IFin mutation carrier (Table 6Down).


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  		Table 6. Activation of LCAT by Different Proteoliposome Substrates

Cholesterol Efflux From Fibroblasts
Human embryonic fibroblasts (HES cells) were loaded with albumin-bound [3H]cholesterol and efflux from the plasma membrane examined using three different acceptor particles: total HDL (from an apoA-IFin carrier or noncarrier), total apoA-I/DMPC discs (apoA-I from an apoA-IFin carrier or noncarrier), and IEF-purified apoA-I/DMPC discs (apoA-I from an apoA-IFin carrier or noncarrier). To clarify whether the apoA-IFin would result in anomalous DMPC binding and subsequent disc formation, acceptor particles were examined by polyacrylamide gradient gel electrophoresis before use. Analysis of the Coomassie brilliant stained gels showed the presence of both apoA-IFin- and wild-type apoA-I-containing particles, and no free apoA-I could be detected (Fig 3Down). All efflux experiments were performed in at least triplicate and repeated at least twice. Initial testing of the HESS cell system using increasing doses of normal apoA-I/DMPC discs resulted in efflux curves showing apoA-I dose dependence (data not shown).



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  		Figure 3. Nondenaturing gradient gel electrophoresis of apoA-I combined with DMPC. Lane 1, apoA-IFin, purified by IEF and combined with DMPC; lane 2, wild-type apoA-I, purified by IEF and combined with DMPC; lane 3, total apoA-I from a control subject combined with DMPC; lane 4, protein standards with diameters (from top to bottom) of 17, 12.2, 10.4, 8.1, and 7.1 nm.

Native HDL preparations isolated from two affected subjects (17 and 18) were compared with total HDL samples from a nonaffected subject (subject 19) and HDL from an unrelated control subject in different doses, and efflux values were counted at 1, 2, 5, 7, 9, and 24 hours of incubation. Efflux values at 2 and 9 hours of incubation are depicted in Fig 4Down. HDL samples from affected subjects were found to be equally effective promoters of cholesterol efflux as were the HDL preparations from a nonaffected family member and a nonrelated control subject (Fig 4ADown). Increasing doses of total apoA-I from an affected and a nonaffected subject combined with DMPC yielded dose-dependent quantities of [3H]cholesterol efflux and showed that apoA-IFin does not impair the promotion of cholesterol efflux from fibroblasts; in fact, apoA-IFin tended to result in slightly higher efflux percentages in comparison with the normal apoA-I (Fig 4BDown). Comparable efflux percentages were also observed when purified preparations of A-IFin or wild-type apoA-I isoform 1 were combined with DMPC and used as acceptors (Fig 4CDown).



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  		Figure 4. Efflux of 3H-labeled cholesterol from human embryonic fibroblasts (HES cells). Efflux was calculated as the percentage of radioactivity present in the media in relation to the initial cellular radioactivity. Values are mean±SE (n=4). A, After loading with [3H]cholesterol, HES cells were incubated for 2 or 9 hours with total HDL fraction (150 µg or 250 µg) isolated from various sources (C, control subject, M, nonaffected family member, P1 and P2, affected subjects). B, After loading with [3H]cholesterol, HES cells were incubated for 2 or 9 hours with total apoA-I (control subject, white bars; affected subject, shaded bars) combined with DMPC. C, After loading with [3H]cholesterol, HES cells were incubated for 7 hours with apoA-I isoforms (5 or 20 µg) isolated via IEF (wild-type apoA-I, white bars; apoA-IFin, shaded bars) and combined with DMPC.


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 *Discussion
down arrowReferences
 
Genetic apoA-I defects provide an unique opportunity to investigate the role of apoA-I in HDL metabolism and to gain knowledge of functionally essential regions in the apoA-I protein. We have examined the impact of apoA-IFin mutation, a single T-to-G transversion at position 2024 of the apoA-I gene (for nucleotide numbering see 42 causing dominantly inherited hypoalphalipoproteinemia, on lipoprotein metabolism in a large kindred with nine affected and nine nonaffected family members in Finland. We have previously shown that A-IFin mutation carriers tend to have slightly elevated serum triglyceride and very low HDL-C and apoA-I concentrations.16 A more profound lipoprotein analysis, carried out in the present study, revealed that the apoA-IFin mutation is associated with several lipoprotein abnormalities, affecting not only HDL but also IDL and LDL particles. In addition, the current study was addressed for clarification of the mechanisms whereby this mutation affects HDL metabolism in vitro and in vivo.

The most prominent feature in the lipoprotein profile of our affected family members was cholesterol-poor and small, dense HDLs, but the metabolic basis for disappearance of larger HDL particles corresponding to the size class of HDL2 is obscure. Hypertriglyceridemia has been proposed to accelerate CETP-mediated exchange of HDL cholesterol esters to triglycerides; triglyceride-rich HDL2 would then be hydrolyzed by hepatic lipase into smaller HDL3 particles. The presence of the CETP transgene in hypertriglyceridemic human apoC-III transgenic mice has actually been shown to greatly reduce HDL particle size.43 CETP activity in the affected subjects was, however, only half of that in the nonaffected subjects and would more likely contribute to the maintenance of larger HDL particles. Elevated serum triglycerides, on the other hand, were present only in five of the mutation carriers, yet all nine mutation carriers exhibited a scarcity of larger HDL2 size particles.

Distribution of serum triglycerides showed a special characteristic with the affected members, ie, a disproportional increment of triglycerides within the LDL fraction, independent of serum total triglyceride level. Only affected member 17, with a low serum total triglyceride concentration, had normal levels of LDL triglycerides (Table 1Up). Enrichment of LDL particles with triglycerides to a various extent has been previously described in different forms of hypoalphalipoproteinemias including Tangier disease,44 hypoalphalipoproteinemia resembling Tangier disease,45 LCAT deficiency,46 and carriers of the A-IMilano mutation.27 Several explanations have been proposed. Triglyceride-rich LDL could reflect an impaired capability of HDL to accept triglycerides because of quantitative and structural abnormalities of HDL particles, lack of exchangeable cholesterol esters, or disturbances of activities with CETP, hepatic lipase, or lipoprotein lipase. In apoA-IFin carriers, the quantitative scarcity of HDL-C may limit the capacity of HDL to accept triglycerides, which are then redistributed onto the LDL fraction.

PLTP enhances the conversion of HDL3 particles to larger HDL2-type particles by a particle fusion mechanism47 and simultaneous formation of small, apoA-I-containing pre-ß type particles.48 The possibility of impaired PLTP functioning in conversion of HDL3 to HDL2 has not been addressed in previous studies on various forms of hypoalphalipoproteinemia. We showed that PLTP in vitro converts apoA-IFin containing HDL3 to HDL2 to the same extent that it converts wild-type HDL3 and that PLTP activity in serum of the affected subjects is normal. These data suggest that the lack of HDL2 is not due to impaired PLTP-mediated conversion mechanisms. The observation that small (pre-ß type) HDL particles were released in the conversion experiments in both the affected and nonaffected subjects (Fig 2Up), together with the previous data showing that pre-ß particles are the initial acceptors of cell-derived cholesterol,49 indicate that apoA-IFin does not alter RCT by interfering with the formation of pre-ß particles, at least via the function of PLTP.

To further investigate the mechanism of the dominant effect of A-IFin on HDL-C and apoA-I levels, apoA-I kinetics was examined in two affected subjects with normal serum triglyceride levels. We have previously shown that the mutant apoA-I reassociates with circulating HDL particles to approximately the same extent as the normal one in postinjection samples, thus permitting the use of the two-pool method in kinetic analysis. In accord with our prior studies, apoA-I FCRs of the affected subjects were higher than those of the control subjects, suggesting that increased catabolism, possibly associated with diminished transport rate, of apoA-I protein could contribute to profoundly diminished HDL-C and apoA-I concentrations (Table 4Up). Hypercatabolism of apoA-I has been observed in a number of inherited disorders, including carriers of apoA-I Milano50 and apoA-IIowa mutations,51 patients with Tangier disease,52 apoA-I deficiency without a detectable apoA-I gene mutation,53 and LCAT deficiency.46 However, increased apoA-I FCR has been reported also in healthy subjects with low HDL-C with or without hypertriglyceridemia.32,52,54 In hypertriglyceridemia, the core of HDL may become rich in triglyceride whereby subsequent lipolysis of HDL core by hepatic lipase may loosen the binding of apoA-I to HDL particle and facilitate clearance of apoA-I by the kidneys.55 Affected family members volunteering for kinetic studies in the present study had both low serum triglycerides, in contrast with the previously studied affected subject 11, yet their FCRs were elevated.

In LCAT deficiencies esterification of cholesterol in the periphery is severely disturbed and maturation of discoid HDL to spherical, cholesterol ester-rich particles is inhibited.56 HDL particles in affected subjects are thus very small in size. Small HDLs occupied almost the whole HDL pool of apoA-IFin carriers, but their LCAT was functioning as cholesterol ester percentages in HDL and LDL were normal and in serum only slightly reduced. However, results from in vitro LCAT activation studies using purified apoA-IFin and artificial proteoliposomes revealed that A-IFin mutant protein diminished LCAT activation to approximately 40% of the normal. The ability of apoA-I to bind phospholipid is a fundamental requirement for LCAT activation. Our in vitro experiments suggest that the ability of apoA-IFin to bind phospholipid is not altered and thus cannot explain the reduction of LCAT activation capacity. It is conceivable that the positive charge difference in apoA-IFin affects either its binding to LCAT or the activation of the enzyme, but the exact mechanism remains unknown. The observation that LCAT activation on interaction with total A-I from affected-subjects was normal implies that apoA-IFin does not interfere with the function of its normal apoA-I counterpart, and half of the normal apoA-I is enough for sufficient LCAT activation in vitro. The total in vivo impact of apoA-IFin on LCAT activation remains obscure and would require a homozygous subject for the apoA-IFin mutation or a transgenic animal model, none of which is available at the moment.

LCAT-activating regions of apoA-I have not yet been fully clarified. Studies using site-directed mutagenesis have indicated that several apoA-I domains are important for LCAT activation, but it is especially the deletion of domains 143 to 164 or 165 to 186 that virtually abolishes LCAT activation by apoA-I.57,58 The conserved region, spanning from 143 to 165 with a pattern of positively charged residues, has been suggested to bind LCAT. In the LCAT molecule, residues 151 to 174 represent a similar region but with a distribution of negative charges, and a molecular model has been presented for residues 151 to 174 of LCAT binding antiparallel to residues 143 to 165 of apoA-I.8 The importance of the region is further enlightened by apoA-ISeattle, a de novo mutation deleting amino acids from Glu146 to Arg160, which diminishes HDL-C apoA-I concentrations to levels below 15% of normal when present in a heterozygous form.17 Furthermore, point mutations at residues 143 (Pro->Arg)59 and 165 (Pro->Arg)60,61 of the apoA-I have been shown to reduce LCAT activation. ApoA-IFin, reducing LCAT activation by 60% in vitro, further underscores the importance of this specific area of apoA-I for interaction with LCAT.

Association between genetic HDL deficiencies and premature CAD, although inevitably an important question, has yet remained obscure. Risk assessment is hindered by the fact that apoA-I and LCAT defects are genetically heterogeneous and rare in the population. In most kindreds with hypoalphalipoproteinemia due to either apoA-I or LCAT deficiency, no clear association of the genetic defect to CAD has been observed. An increased propensity for CAD has been demonstrated in specific conditions, such as homozygosity for the apoA-I Gln 84->Stop or Q(-2)X mutations14,21 or major rearrangements of the apoA-I/C-III/apoA-IV gene complex.62,63 An Italian family with hypoalphalipoproteinemia due to compound heterozygosity for an apoA-I null allele and apoA-IL 141 R Pisa mutation was recently described.15 Several mutation carriers suffered from clinically manifested CAD, but they also exhibited many additional risk factors such as hypercholesterolemia, obesity, hypertension, and smoking. Eight of the nine carriers of the apoA-IFin mutation were devoid of any clinical signs of CAD despite their very low serum HDL-C levels. Their increased CAD risk cannot be excluded due to the relatively small sample size and young age of five affected family members. However, family member 16 had undergone a coronary bypass operation at the age of 60 and had no other risk factors for CAD except the low HDL due to apoA-IFin mutation. In some studies, low HDL-C has been shown to associate with the occurrence of small, dense LDLs widely considered to be atherogenic.64 Lipoprotein analysis of apoA-IFin carriers did not disclose any evidence for increased amounts of small, dense LDL particles in their serum.

In view of the proposed role of HDL particles as cholesterol acceptors in RCT, we examined the influence apoA-IFin mutation on cholesterol efflux from plasma membranes using in vitro fibroblast cell culture. Comparative efflux experiments with purified A-IFin and their wild type counterpart particles in vitro suggest that the A-IFin mutation does not cause conformational changes interfering with the putative interaction of apoA-I with plasma membrane and passive diffusion of cholesterol from the plasma membrane to the acceptor particles. The domains necessary for cholesterol efflux have not been thoroughly identified and may actually depend on the cell type and particle type used. Thus, apoA-I amino acid residues 74 to 111 were found to be critical for cholesterol efflux from monocytes by monoclonal antibodies,65 but the epitope associated with the efflux of intracellular cholesterol from HepG2 cells was shown to correspond to residues 140 to 15066; the latter epitope was not, however, critical for efflux from plasma membrane, which was the main cholesterol reservoir labeled in the present study. Furthermore, the apoA-I Pro165Arg mutation was shown to slightly decrease cholesterol efflux from macrophages using apoA-I/DMPC particles.41 Comparison of different efflux experiments is hampered by the fact that several factors account for the actual cholesterol efflux in vitro,8,67 and different cell types, labeling methods, and incubation times as well as heterogeneous acceptor particles (size, density, and lipid composition) have been used.

Experiments in which total HDLs are used as acceptors may represent the situation closest to that in vivo. Our results indicate that HDLs from affected members are at least as efficient promoters of cholesterol efflux in vitro as are the HDLs from nonaffected members. Studies with serum of transgenic rats overexpressing human apoA-I have proposed that the main factor determining the cholesterol acceptor capacity would be the concentration of HDL phospholipids.68 Because the ratio of HDL phospholipid to protein is significantly increased in apoA-IFin carriers compared with noncarriers (1.1 versus 0.75, respectively, P<.001) and the amount of total HDLs used in efflux experiments were standardized according to their protein concentration, the higher amounts of phospholipids present in A-IFin HDL acceptor particles might have contributed to in vitro efflux-promoting capacity. On the other hand, protein composition of apoA-IFin HDL shows a disproportionally high amount of apoA-II which has in some,69,70 but not all,71,72 studies shown to inhibit cholesterol efflux depending on the cell type and the cellular location (plasma membrane or intracellular pool) of the cholesterol for efflux.73,74 Therefore, the disproportionally high amount of apoA-II in the HDL might not have affected cholesterol efflux from the plasma membrane in the present study protocol although the situation in vivo may be different. Indeed, apoA-II transgenic mice were shown to be vulnerable to diet-induced atherosclerosis,75 and animals who have apoA-I/apoA-II HDL predominating over apoA-I HDL were at greater risk of aortic cholesterol accumulation.76

Interpretation of the efflux results in vitro concerning the actual situation in vivo and the possible atherogenicity of apoA-IFin is hindered by several factors. Despite extremely low HDL apoA-I levels, even trace amounts of normally functioning apoA-I protein may be sufficient for the process of RCT in vivo or that factors other than apoA-I, such as apoE-containing plasma lipoprotein {gamma}-LpE,77 can also trigger cholesterol efflux from cells. It should also be emphasized that the protective effect of HDL against CAD may occur also via processes other than RCT, such as protection of plasma lipoproteins from peroxidation78 or promotion of fibrinolysis,79 and the role of apoA-IFin in these events remains to be elucidated.

In conclusion, heterozygosity for the apoA-IFin mutation results in severe reduction of serum HDL-C concentration, a virtual lack of large HDL2-size particles, and enhanced catabolism of apoA-I. In vitro studies using reconstituted proteoliposomes showed a decreased capacity of apoA-IFin to activate LCAT but an apparently intact ability to promote cholesterol efflux from fibroblasts. Elucidation of the atherogenic or antiatherogenic potential, if any, of apoA-IFin still requires further studies in additional affected kindreds and in transgenic animals.


*    Selected Abbreviations and Acronyms
 
apo = apolipoprotein
CAD = coronary artery disease
CETP = cholesteryl ester transfer protein
DMPC = dimyristoyl phosphatidylcholine
FCR = fractional catabolic rate
HDL-C = high-density lipoprotein cholesterol
HESs = human embryonic fibroblasts
IEF = isoelectric focusing
LCAT = lecithin:cholesterol acyltransferase
PAGE = polyacrylamide gel electrophoresis
PC = phosphatidylcholine
PCR = polymerase chain reaction
PLTP = phospholipid transfer protein
RCT = reverse cholesterol transport
SDS = sodium dodecyl sulfate
TBS = Tris-buffered saline


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  		Table 1A. Continued


*    Acknowledgments
 
Susanna Tverin, Merja Lindfors, Leena Saikko, and Ritva Keva provided excellent technical assistance. This study was supported by grants from the Medical Council of the Finnish Academy, The Sigrid Juselius Foundation, Finnish Heart Foundation, the Paulo Foundation, Orion Research Foundation, Finnish Medical Society Duodecim, and the University of Helsinki.


*    Footnotes
 
Revision received April 7, 1997; revision accepted June 27, 1997.


*    References
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 *References
 

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