© 1995 American Heart Association, Inc.
Articles |
Effect of Glycation on the Properties of Lipoprotein(a)
From the Departments of Medicine, Biochemistry (A.M.S.), and Molecular Biology (A.M.S.), University of Chicago, Chicago, Ill.
Correspondence to Dr Gunther M. Fless, Department of Medicine, MC 5041, The University of Chicago, 5841 S Maryland, Chicago, IL 60637.
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Abstract Lipoprotein(a) [Lp(a)] was glycated by incubation in vitro with glucose (0 to 200 mmol/L), and its properties were compared with native Lp(a) and native and glycated LDL. Glucose was incorporated into Lp(a) in proportions that mirrored the distribution of lysines between apolipoprotein (apo) B-100 and apo(a). Because the kringle IV domains of apo(a) are lysine poor, only 10% of glucose bound to apo(a), whereas 90% was attached to the apoB-100 of Lp(a). Approximately 3% of the lysines of both Lp(a) and LDL were modified, which is a level comparable with that observed in LDL isolated from diabetic individuals. Glucose uptake by Lp(a) and LDL was almost identical and was linear as a function of concentration and time. Glycation increased the negative charge of Lp(a) and LDL as monitored by electrophoresis and ion-exchange chromatography and also reduced the affinity of Lp(a) and LDL for heparin-Sepharose. Glycation did not affect the lysine-binding property of Lp(a) or generate measurable malondialdehyde oxidation adducts. The catabolism of glycated Lp(a) by human monocyte–derived macrophages (HMDMs), like that of native Lp(a), was largely LDL receptor independent. Both glycated Lp(a) and LDL were degraded at a comparatively faster rate and stimulated greater cholesteryl ester formation than their unmodified counterparts. However, the degradation rate of glycated Lp(a) was approximately four- to fivefold slower and its stimulation of cholesteryl ester formation was ninefold lower than that of either form of LDL. These results show that Lp(a) can be glycated nonenzymatically in vitro, that the incorporation of glucose is dependent on the distribution of lysines between apo(a) and apoB-100, and that glycation does not affect the lysine-binding properties of Lp(a). Furthermore, glycation produced modest increases in the degradation rate of Lp(a) and associated cholesteryl ester synthesis by HMDMs. Based on these data, glycation does not appear to significantly enhance the atherogenic potential of unmodified Lp(a).
Key Words: diabetes • lysine binding • charge • macrophages • degradation
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Epidemiological studies have implicated high levels of plasma lipoprotein(a) [Lp(a)] with increased risk for atherosclerotic cardiovascular disease.1 Since atherosclerosis is a major complication associated with diabetes, much effort has been spent on elucidating the possible role of Lp(a) in this disease.2 3 4 5 6 7 8 9 10 11 12 13 14 Glycation of proteins, ie, nonenzymatic glycosylation resulting from the high plasma glucose levels found in diabetes, is thought to be one of the factors contributing to the severity of this disease.15 All major apolipoproteins, including apolipoprotein (apo) B-100 of LDL, usually have some of their lysine residues modified by nonenzymatic glycosylation.16 The functional consequences of glycation are manifested in impaired LDL receptor recognition of glycated LDL in fibroblasts and human monocyte–derived macrophages (HMDMs)17 18 and increased cholesterol ester synthesis in HMDMs, possibly via a high-capacity, low-affinity, non-LDL receptor pathway.18 Glycation of apoB-100 also slows the catabolism of LDL.19
Although the physical and metabolic properties of in vitro glycated LDL have been studied at length, similar investigations of Lp(a) have not been performed. In the present study we used Lp(a) that was glycated in vitro to examine its interaction with lysine- and heparin-Sepharose. Additionally, we studied the metabolic consequences of glycation in HMDMs in terms of Lp(a) degradation and cholesterol ester synthesis in comparison with native Lp(a). In all studies native and glycated LDL served as controls.
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Materials
Sodium [125I]iodide, [U-14C]glucose, [1-14C]oleic acid, and [1,2-3H]cholesteryl oleate were purchased from Amersham Corp; Dulbecco's modified Eagle's medium (DMEM) containing 25 mmol/L HEPES, L-glutamine, and 4500 mg/L glucose, and phosphate-buffered saline (PBS) minus calcium and magnesium were from GIBCO Laboratories; Plasmagel was from Cellular Products Inc; phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, triolein, oleic acid, cholesteryl oleate, bovine serum albumin fraction V, and human serum albumin (HSA) were all purchased from Sigma Chemical Co; and Ficoll-Hypaque and PD-10 Sephadex G-25 columns were from Pharmacia. Plastic 12-well tissue-culture dishes were from Flow Laboratories, and heparin USP 1000 U/mL was obtained from LyphoMed.
Preparation of Lp(a) and LDL
Two healthy subjects, one man (age, 36 years; Lp(a) protein
level, 23 mg/dL) and one woman (age, 42 years; Lp(a) protein level, 32
mg/dL) donated plasma on a bimonthly basis after giving informed
consent. Autologous LDL and Lp(a) were isolated from their
plasma.20 Both Lp(a) species had a fast apo(a) polymorph
with a molecular weight of 281 00021 and were assumed to
behave equivalently. The molecular mass of the protein moiety of this
Lp(a) was determined previously from the molecular mass and chemical
composition of Lp(a) and found to be 918 000 D.21
Lipoproteins were freshly prepared on a monthly basis and were usually
used before 1 month had expired. Lp(a) and LDL in 0.15 mol/L NaCl,
0.01% Na2-EDTA, and 0.01% NaN3, pH
7.4, when kept sterile in Sarstedt tubes at 4°C that are filled to
the top to minimize air space, exhibit no degradation, oxidation, or
loss of immunochemical activity.22
Electrophoretic Methods
Lipoprotein purity was determined by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis on 2% to 16%
polyacrylamide gels (IsoLab).23 Electrophoresis on precast
1% agarose films (Corning ACI) using a Corning unit for 35 minutes was
performed according to the manufacturer's instructions. The films were
stained with Coomassie blue R-250.
Chromatographic Methods
Heparin (type II) agarose chromatography was performed by
using the low-pressure chromatography BioRad Econo System (BioRad
Laboratories). The column (6 cmx0.2 cm2) was equilibrated
with 0.02 mol/L NaCl, 0.01% Na2-EDTA, and 0.01%
NaN3 and was monitored at 280 nm. Lipoproteins (200 µg
protein) equilibrated in the same buffer were applied to the column,
washed with equilibration buffer, and eluted with a linear gradient of
0.02 to 1.0 mol/L NaCl. The flow rate was 10 mL/h, and 0.5-mL fractions
were collected. The salt content of individual fractions was determined
by refractometry; protein content was determined by measuring the
absorbance at 280 nm.
Ion-exchange chromatography of the lipoproteins was performed by using a MonoQ column and a fast protein liquid chromatography system (Pharmacia).24 Lipoprotein (200 µg) was applied to the MonoQ column in 10 mmol/L Tris, 0.01% Na2-EDTA, and 0.01% NaN3, pH 7.4, at 8°C. The lipoproteins were eluted with a linear gradient consisting of 10 mmol/L Tris, pH 7.4, and 10 mmol/L Tris and 0.5 mol/L NaCl, pH 7.4. The absorbance was monitored at 280 nm.
Lysine-Sepharose chromatography of native and glycated Lp(a) was
performed on a 1-mL minicolumn (5 cmx0.2
cm2).24 Aliquots of Lp(a) (200 µg)
equilibrated with 0.15 mol/L NaCl, 10 mmol/L phosphate, 0.01%
Na2-EDTA, and 0.01% NaN3, pH 7.4, were
applied to lysine-Sepharose minicolumns that were equilibrated in the
same buffer. Bound Lp(a) was eluted from the column with 200 mmol/L
-amino-n-caproic acid dissolved in equilibration buffer.
The column was monitored at 280 nm by using an absorbance monitor
(model 150, Altex Scientific, Inc) equipped with a chart recorder
(model BD 41, Kipp/Zonen).
Glycation of Lp(a) and LDL
Glycation of Lp(a) and LDL was performed by incubating Lp(a) and
LDL (1 mg/mL) with varying concentrations of glucose in sterile PBS
containing 1 mmol/L EDTA (pH 7.4) at 37°C for 7 days. The degree of
glycation of Lp(a) and LDL was measured by enzyme-linked immunosorbent
assay (ELISA) after affinity chromatography with
m-aminophenylboronate gel (Glycogel B, Pierce). Minicolumns
containing 1 mL of the gel were prepared and equilibrated with 5 mL of
250 mmol/L ammonium acetate, 50 mmol/L MgCl2, 500
mmol/L NaCl, 3 mmol/L NaN3, and 0.1% Tween-20, pH
8.5, at 20°C. A 200-µL aliquot of incubation mixture was added and
allowed to soak into the gel, followed by 20 mL of equilibration
buffer. The collecting vessel was changed, and the glycated lipoprotein
fraction was eluted from the gel column by adding 3 mL of 200 mmol/L
sorbitol, 500 mmol/L NaCl, 50 mmol/L Na2-EDTA, 100 mmol/L
Tris, and 0.1% Tween-20, pH 8.5. The concentration of Lp(a) or LDL in
the glycated and nonglycated fractions was determined by ELISA
according to the method described by Fless et al22 and
expressed as a percentage of total lipoprotein.
Assays of Chemical Modification
The number of lysine residues modified in lipoproteins upon
glycation was estimated with the trinitrobenzenesulfonic acid
assay.25 Products of lipoprotein oxidation in terms of
malondialdehyde (MDA) adducts were determined by the thiobarbituric
acid colorimetric assay at 532 nm.26
Iodination of Lp(a) and LDL
The radioiodination of Lp(a) and LDL was performed by using the
iodine monochloride method of McFarlane27 as modified by
Bilheimer et al28 and described by us.20
Specific radioactivities for both lipoproteins averaged 600 cpm/ng
lipoprotein protein.
Isolation and Culture of HMDMs
Isolation of human monocytes was performed essentially as
reported by Fogelman et al29 and described by
us.20 The monocytes were suspended in DMEM (20%
autologous serum) at a final concentration of
1.0x106 cells/mL; 1 mL was plated into each well of
Linbro 12-well tissue-culture plates (area per well, 4.5
cm2). The medium was replaced on the second and fifth days
of culture for 8-day-old cells and on the second, fifth, and eighth
days for 10-day-old cells. Twenty-four hours prior to the initiation of
experiments, the cells were placed in DMEM containing either 20%
autologous lipoprotein-deprived serum (LPDS) or 20% autologous serum.
The cells were greater than 99% monocyte-macrophages by the time the
experiments were performed due to the frequent washes and medium
changes.
Cholesterol Esterification Assays
Cholesteryl[14C]oleate formation in HMDMs was
determined essentially as described by Innerarity et al.30
Briefly, monolayers of macrophages that had been upregulated for 24
hours in 20% autologous LPDS were washed twice with serum-free DMEM
and then incubated for 16 hours at 37°C with 0.2 mmol/L
[14C]oleate/albumin (7200 dpm/nmol) in the presence of
100 or 200 nmol/L of either Lp(a) or LDL. After incubation, the
monolayers were washed, and the lipids containing cholesteryl esters
were extracted with hexane-isopropanol (3:2) and isolated by thin-layer
chromatography. The cholesteryl[14C]oleate was quantified
by liquid scintillation counting with [3H]cholesteryl
oleate as internal standard. After the lipids were extracted, the cells
were dissolved in 0.1 mol/L NaOH for protein determination. The
cellular protein was determined by the method of Lowry et
al31 as modified by Markwell et al32 using
bovine serum albumin as standard.
37°C Degradation Assays
Degradation experiments were performed essentially as described
by Goldstein et al.33 Briefly, the cell monolayers were
washed once with DMEM and then twice with DMEM (0.2% HSA). Either
125I-Lp(a) or 125I-LDL (100 nmol/L each) in
DMEM containing 2 mg/mL HSA was added to the cells, and the cells were
returned to the 37°C incubator for 5 hours. The proteolytic
degradation of the 125I-labeled lipoproteins was measured
by assaying the amount of 125I-labeled trichloroacetic
acid–soluble (noniodide) material formed by the cells and excreted
into the medium. The cellular protein was determined as described for
the cholesterol esterification study.
Statistics
The statistical significance of differences between means was
estimated by using either the paired or unpaired Student's
t test. Analyses were performed using the software program
STATVIEW.
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Glucose incorporation into Lp(a) was investigated as a function of time by incubating 0.2 mg/mL of Lp(a) protein (0.2 µmol/L) with 200 mmol/L glucose in PBS and 1 mmol/L EDTA, pH 7.4, at 37°C under sterile conditions. LDL served as control. The observed time course of glucose incorporation (Fig 1
, left), as measured with
boronate gel affinity chromatography, was nearly identical for both
lipoproteins. Each exhibited a lag phase of 48 hours before glucose
incorporation increased linearly to values of 17% glycation after a
7-day incubation period. The degree of glycation of either lipoprotein
was directly related to the glucose concentration in the incubation
mixture and was again similar for both lipoproteins (Fig 1, right).
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Because the possibility existed that Lp(a) and LDL might become prone to oxidation when incubated in the presence of glucose at the relatively high temperature of 37°C, we determined oxidation by measuring MDA adducts of both glycated (200 mmol/L glucose, 37°C, 7 days) and control lipoproteins with the thiobarbituric acid assay. Endogenous MDA was extremely low in both native Lp(a) and LDL, and the concentrations did not increase with glycation.26 Both native and glycated Lp(a) had less than 0.4 mol MDA/mol, whereas native and glycated LDL had less than 0.2 mol MDA/mol lipoprotein.
The distribution of [14C]glucose between apo(a) and
apoB-100, the two constitutive proteins of Lp(a), was also determined.
Freshly prepared [14C]glucose (12.1 dpm/pmol glucose) at
a concentration of 5 mmol/L was incubated with 2 µmol/L Lp(a) in PBS
and 1 mmol/L EDTA, pH 7.4, under sterile conditions at 37°C for 7
days. Apo(a) was separated from the parent 14C-glycated
Lp(a) after reduction of Lp(a) with 50 mmol/L dithiothreitol and
rate-zonal density gradient centrifugation.20 21 The
floating lipoprotein particles, containing apoB-100 but not apo(a), and
apo(a) from the centrifuge tube bottom were counted for radioactivity
and corrected for the radioactivity found in the lipid moiety. Only
13% of the incorporated [14C]glucose appeared in apo(a),
whereas apoB-100 accounted for 87% (see Table 1). These
values corresponded well with the distribution of lysines in the two
apoproteins as calculated from their respective amino acid sequences,
which were 11.4% for apo(a) and 88.6% for apoB-100.
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All further studies were performed with samples of Lp(a) and LDL that
were glycated with 200 mmol/L glucose for 7 days at 37°C. The actual
percentage of modified amino groups in the two glycated lipoproteins
was assessed with the trinitrobenzenesulfonic acid assay. The number of
amino groups modified by 200 mmol/L glucose was 3.1% in Lp(a) and
2.6% in LDL. Although modification of amino groups was small, the
increased electronegativity of the glycated lipoproteins could be
detected by agarose gel electrophoresis. Both glycated lipoproteins
showed slightly increased anodic migration in contrast to the native
Lp(a) or LDL that was incubated under the same conditions in the
absence of glucose (Fig. 2).
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The increased electronegativity of the glycated lipoproteins could also
be detected by fast protein liquid chromatography by using a MonoQ
column (Pharmacia). Glycated Lp(a) had a small but significantly
greater affinity (4%) for the anion-exchange resin MonoQ than native
Lp(a) incubated at 37°C for 7 days (Table 2). Glycated
LDL also had a greater affinity (5.6%) than the heated native LDL
control. Both glycated lipoproteins had symmetrical elution peaks and
did not exhibit heterogeneity upon glycation.
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Glycation of Lp(a) also reduced its affinity for heparin-Sepharose in
that the concentration of NaCl necessary to release the lipoprotein
from the column decreased from 0.13 to 0.1 mol/L (Fig 3). A similar decrease was observed with glycated LDL,
which eluted from the column with 0.2 mol/L NaCl, in comparison with
native LDL, which emerged with 0.24 mol/L NaCl. Both forms of Lp(a) had
substantially lower affinities for heparin-Sepharose than their LDL
counterparts.
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The effects of glycation on the ability of Lp(a) to bind to
lysine-Sepharose were also investigated. Aliquots (200 µg protein) of
native Lp(a), Lp(a) incubated at 37°C, and glycated Lp(a), all
equilibrated with 0.15 mol/L NaCl, 10 mmol/L phosphate, 0.01%
Na2-EDTA, and 0.01% NaN3, pH 7.4, were
applied to 1-mL lysine-Sepharose minicolumns that were equilibrated in
the same buffer. In each case, all the lipoprotein bound to the column
and could be eluted with 200 mmol/L -amino-n-caproic acid
in equilibration buffer. Glycation of Lp(a) therefore did not affect
its ability to bind to lysine-Sepharose.
The consequences of in vitro glycation on the interaction of Lp(a) or
LDL with HMDMs were assessed by studying its effect on the degradation
of Lp(a) and LDL and on cholesterol esterification. Total degradation
by upregulated HMDMs, which includes both LDL-specific and
LDL-nonspecific components, was increased significantly with either
glycated Lp(a) or LDL (Fig 4). At 100 nmol/L
radioiodinated lipoprotein, the degradation rate of Lp(a) increased
from 0.41±0.03 to 0.53±0.03 pmol · 5
h-1 · mg-1 cell protein
(P<.0005) in response to glycation, which
represents a 29% increase. A somewhat smaller elevation of
21% was observed with glycated LDL, which increased from 1.89±0.18 to
2.28±0.20 pmol · 5 h-1 · mg-1 cell
protein (P<.0005). Overall, the degradation rates of both
native and glycated Lp(a) were only 21% to 23% of the corresponding
rates attained with LDL.
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The importance of the LDL receptor pathway in the degradation of native and glycated Lp(a) was assessed by downregulating HMDMs with a preincubation in 20% whole human serum. The degradation rate of glycated Lp(a) decreased by only 13% and that of native Lp(a) by 22% when comparing downregulated with upregulated HMDMs, indicating that LDL receptor–mediated degradation of both native and glycated Lp(a) is very low compared with other degradation pathways. Glycation of Lp(a) increased its degradation rate by downregulated HMDMs from 0.32±0.03 to 0.46±0.03 pmol · 5 h-1 · mg-1 cell protein (P<.0005 versus control by paired Student's t test), which is an increase of 43% (mean±SD of quadruplicate determinations of four experiments with two different sets of lipoproteins from each donor).
Glycation of Lp(a) caused a significant increase (37.5%) in the
rate of cholesterol esterification in HMDMs when compared with native
Lp(a) (0.022±0.002 versus 0.016±0.001
nmol · h-1 · mg-1 cell protein; Fig 5). A similar increase of 36.6% was observed when
glycated LDL was compared with the native LDL control (0.198±0.032
versus 0.145±0.014 nmol · h-1 · mg-1
cell protein). However, both native and glycated Lp(a) stimulated only
11% as much cholesterol ester synthesis as was achieved with the
corresponding LDL fraction.
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These results indicate that the effects of in vitro glycation on the physical and functional properties of Lp(a) and LDL are similar, despite the fact that Lp(a) contains a second apolipoprotein, apo(a), in addition to apoB-100. Considering that glucose forms adducts mainly with the
-amino group of lysine residues,34 of which
almost 90% are contained in apoB-100, this finding is perhaps not very
surprising. The unusual distribution of lysine residues between apo(a)
and apoB-100 is caused by their limited abundance in the kringle IV
domains, which make up the major portion of apo(a).35
Specifically, the kringle V and protease domains of apo(a) contain 20
lysine residues, kringles IV-9 and IV-4 each have one, and the
interkringle region between kringles IV-8 and IV-9 has an additional
lysine residue. The distribution of lysine between apo(a) and apoB is
not subject to polymorphism because all apo(a) polymorphs have the same
number of lysines, which are located in the invariable domain of
apo(a). Because the lysine content was almost the same, nearly identical curves were obtained with both lipoproteins when the amount of glucose incorporated into Lp(a) and LDL as a function of time and glucose concentration was compared on a molar basis. Approximately 3% of the lysine residues of either Lp(a) or LDL were modified by glucose, which is at a level comparable with that observed in LDL isolated from diabetic individuals.19 It must be emphasized, however, that the in vitro glycation as performed under the present conditions (ie, no air space and in the presence of EDTA) minimized oxidation, as shown by the fact that neither Lp(a) nor LDL had measurable oxidation products in the form of MDA adducts. This contrasts with in vivo conditions, under which increased glycation may induce oxidative damage in proteins with time.34
Although native Lp(a) has properties distinctly different from those of native LDL, glycation produced equivalent changes in both lipoproteins. Thus, both glycated lipoproteins had similar increases in mobility on agarose gels and affinity for the anionic-exchange resin MonoQ, which probably resulted from the increased negative charge caused by the modification of lysine amino groups. Additionally, both glycated lipoproteins had a decreased affinity for heparin-Sepharose that was most likely due to the modification of integral lysine amino groups of heparin-binding sites located on apoB-100. The increased electrophoretic mobility and the decreased affinity of glycated LDL for heparin-Sepharose are in agreement with the findings of Witztum et al36 and Gugliucci-Creriche and Stahl,37 respectively.
Since both the heparin- and LDL receptor–binding domains of apoB-100 have positively charged basic regions rich in lysine that appear to overlap (for review, see Reference 3838 ), the reduced affinity for heparin may also indicate a reduced affinity for the LDL receptor. Indeed, decreased recognition of glycated LDL by the LDL receptor of fibroblasts36 39 40 and macrophages18 has been documented, and it has been suggested that the slowed catabolism would increase the residence time and plasma levels of glycated LDL. Although LDL-specific degradation of glycated LDL was diminished, total degradation was increased by macrophages, implying the existence of a separate, low-affinity, high-capacity receptor pathway.18
Our results confirm our previous finding that LDL receptor–mediated degradation of Lp(a) by HMDMs is very low20 41 and show that it is not significantly affected by glycation. Although the affinity of glycated Lp(a) for heparin-Sepharose was decreased, we did not observe a parallel decrease in LDL receptor–mediated degradation of Lp(a), probably because both native and glycated Lp(a) were degraded mainly via nonspecific pathways. However, it is important to note that HMDMs actually degraded significantly more glycated Lp(a) per unit time than native Lp(a), thereby causing an increase in cholesterol ester synthesis. If we can extrapolate the results obtained with HMDMs to the situation existing in vivo, in which glycation of LDL is thought to slow its catabolism because of impaired LDL receptor uptake, the catabolism of Lp(a) may actually be greater in subjects with diabetes because its uptake is largely LDL receptor independent. The recent finding of Rainwater et al42 that the concentration of Lp(a) in patients with non–insulin-dependent diabetes mellitus (NIDDM) is actually lower than in nondiabetic subjects would agree with this hypothesis.
When the degradation rate or stimulation of cholesteryl ester synthesis by native or glycated Lp(a) is compared with that of native or glycated LDL, it is apparent that both forms of Lp(a) are metabolized much more slowly by HMDMs than are their LDL counterparts. Since the uptake of neither native nor glycated LDL by HMDMs is large enough to stimulate foam cell formation by macrophages,18 43 in vitro glycated Lp(a) probably behaves similarly, particularly when one considers that it causes a much lower rate of cholesteryl ester formation than either form of LDL. It is important to point out that the present study considered an Lp(a) species having only the "fast" apo(a) polymorph, and it is unclear if these results can be applied equally to other Lp(a) species.
The exact role played by Lp(a) in the development of coronary artery disease by diabetic patients is unclear (for review, see Reference 4444 ). Recent studies suggest that Lp(a) concentrations are not significantly affected in patients with insulin-dependent diabetes mellitus (IDDM)10 45 or patients with NIDDM42 (in whom Lp(a) levels may actually be lower) compared with nondiabetic control subjects. Results from at least one prospective study of a young cohort of IDDM individuals show that Lp(a) concentrations show little relation to the development of complications.46 In addition, these investigators did not see higher Lp(a) concentrations in patients with overt nephropathy or microalbuminuria, in contrast to other studies that have reported increased Lp(a) levels.4 5 Whether Lp(a) is a risk factor for coronary artery disease in IDDM or NIDDM remains to be established by appropriate prospective studies that also take into account apo(a) polymorphism and Lp(a) density heterogeneity.44 46
Our results may provide a partial explanation for the apparent lack of contribution by Lp(a) to the development of coronary artery disease in IDDM or NIDDM. It is thought that part of the atherogenicity of Lp(a) may be explained by its affinity for lesion-localized fibrin and for sulfated mucopolysaccharides of the extracellular matrix.47 Much of the binding of Lp(a) by fibrin is lysine mediated, and as the present results show, the lysine-binding properties of Lp(a) are not affected by in vitro glycation as performed under these conditions. This is probably caused by the fact that the kringle IV37 domain of apo(a), which is thought to be responsible for lysine binding, has no lysine residues and is therefore not glucosylated, a process that might affect its ligand-binding properties. If, however, a sufficient number of fibrin-lysine residues became glycated as a result of diabetic complications, then it is conceivable that the affinity of Lp(a) for fibrin could be reduced. Additionally, if the interaction of Lp(a) with heparin mirrors its binding behavior to sulfated polysaccharides of the extracellular matrix, then glycated Lp(a) would also have a reduced affinity. Thus, two mechanisms that would localize Lp(a) to regions of lesion development could potentially be diminished by glycation. Whether such a simplistic mechanism, which does not take into account oxidation or other abnormal metabolic factors associated with diabetes, can actually explain the apparent lack of Lp(a) involvement in diabetic atherogenesis remains to be seen.
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Acknowledgments |
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This work was supported by National Heart, Lung, and Blood Institute program project grant HL-18577. The authors gratefully acknowledge Margaret L. Snyder for help in the initial isolation and maintenance of human monocytes.
Received September 23, 1994; accepted December 12, 1994.
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