© 1995 American Heart Association, Inc.
Articles |
Immunological Evidence for the Presence of Advanced Glycosylation End Products in Atherosclerotic Lesions of Euglycemic Rabbits
From the Department of Medicine, University of California, San Diego, La Jolla, Calif (W.P., S.W.B., E.M., J.L.W.); the Diabetes Research Institute, University of Düsseldorf, Germany (T.K.); and The Picower Institute for Medical Research, Manhasset, NY (H.V., A.C.).
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract Atherosclerosis is known to be accelerated in diabetic patients, but the mechanisms of this acceleration are poorly understood. Nonenzymatic glycosylation of long-lived proteins results in the formation of advanced glycosylation end products (AGEs), which are extensively cross-linked and could contribute to atherogenesis. Oxidative modification of LDL is also an important process in atherogenesis. In vitro evidence suggests that hyperglycemia may enhance lipid peroxidation, and conversely, that increased lipid peroxidation may enhance AGE formation. If such interactions occur in vivo, we hypothesized that AGE should be found in atherosclerotic lesions of euglycemic LDL receptor–deficient rabbits in areas rich in lipids and oxidized lipoproteins. To demonstrate the presence of AGEs, we developed antisera against a specific "model" compound of AGE, 2-furoyl-4(5)-(2-furanyl)-1H-imidazole (FFI) by using FFI–hexanoic acid (FFI-HA)–protein adducts as the antigen and against AGEs in general by using AGE-albumin as the antigen. Antisera generated with FFI-HA–protein adducts recognized FFI-HA alone as well as FFI-protein adducts. Native proteins or proteins conjugated with aldehydes formed during lipid peroxidation in vitro were not recognized by these antisera. Immunocytochemistry with both FFI-specific and AGE-specific antisera revealed the presence of these epitopes in atherosclerotic lesions of euglycemic LDL receptor–deficient rabbits but not in normal aortic tissues. AGE epitopes within atherosclerotic lesions were predominantly found in similar locations as epitopes generated during modification of the lipoproteins by oxidation, consistent with the hypothesized interactions between oxidation and glycosylation. Indirect evidence in support of the in vivo presence of FFI-like structures was also obtained by the observation that both diabetic and euglycemic human subjects contained autoantibodies that recognize FFI-protein adducts. Taken together, these data provide immunological evidence for the in vivo presence of FFI-like structures and other AGE-protein adducts in atherosclerotic lesions, even in euglycemic conditions.
Key Words: advanced glycosylation end products • arteriosclerosis • immunocytochemistry • oxidation • autoantibodies
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Vascular pathology represents one of the major long-term complications of diabetes mellitus in both the microcirculation and major vessels (in the form of atherosclerosis). Although epidemiological studies have demonstrated that arteriosclerosis is enhanced by diabetes,1 the mechanisms by which this occurs are not known. Nonenzymatic glycosylation (NEG) of proteins by reducing sugars leads to the formation of advanced glycosylation end products (AGEs).2 3 4 5 6 7 By interfering with functionally important lysine groups and amino-terminal amines, AGE formation has the potential to significantly affect biological and structural properties of proteins and has been postulated to contribute to the progression of many diabetic complications.
Both NEG and AGE formation could enhance atherogenesis by several mechanisms. NEG of LDL and other lipoproteins8 9 could alter lipoprotein metabolism.10 11 In addition, AGE modification has also recently been demonstrated in circulating lipoproteins.12 In the vascular wall, formation of AGE adducts may interfere with vasodilation either by the reduced elasticity of AGE-modified structural proteins2 3 or by interference of AGEs with the vascular response to nitric oxide.13 AGE formation may trap LDL or immunoglobulins in the intima by cross-linking to structural proteins.14 15 In turn, the prolonged intimal residence time of LDL may enhance the generation of oxidized LDL (OxLDL), which may contribute to atherogenesis by multiple mechanisms,16 17 18 including rapid uptake of OxLDL via scavenger receptors.19 Macrophages also possess specific receptors that recognize AGE proteins,20 21 22 23 24 and uptake of AGE-modified lipoproteins via these receptors could contribute to foam cell formation. AGEs could also enhance the intimal accumulation of macrophages either by binding to specific receptors on endothelial cells24 or by a direct chemotactic effect25 26 similar to that of OxLDL.27 Finally, by binding to one of the AGE receptors, AGE-modified proteins may induce endothelial cells to generate a pro-oxidant state.28
A number of in vitro studies suggest that lipid peroxidation and NEG/AGE formation involve some similar intermediates. In vitro, hyperglycemia enhances lipid peroxidation, and conversely, increased lipid peroxidation could augment AGE formation.12 29 30 31 32 33 34 35 If this mutual enhancement also takes place in vivo, it would imply that accelerated formation of AGE products should occur in atherosclerotic lesions of diabetic subjects. Indeed, enhanced lipid peroxidation in general36 37 and AGE formation in particular12 have been demonstrated in circulating lipoproteins of diabetic subjects, and AGE formation in atherosclerotic tissues of diabetics has been demonstrated by immunocytochemistry.38 However, it is not possible to estimate if and how much enhanced lipid peroxidation contributes to increased AGE formation in diabetic subjects, as AGE formation may simply be a direct consequence of hyperglycemia. To address this question, we hypothesized that enhanced AGE formation may also occur in euglycemic Watanabe heritable hyperlipidemic (WHHL) rabbits, which are characterized by extreme hypercholesterolemia. The atherosclerotic lesions of these rabbits are rich in OxLDL,39 40 41 42 and antioxidant therapy can inhibit atherosclerosis in this animal model.16 17 Therefore, the extensive lipid peroxidation that occurs in their lesions should enhance AGE formation, and their atherosclerotic lesions should contain significant amounts of AGEs in contrast to normal rabbit aortas.
To test the hypothesis that products of AGEs would be formed in
atherosclerotic lesions of euglycemic WHHL rabbits, we developed
antisera against a specific compound of AGE,
2-furoyl-4(5)-(2-furanyl)-1H-imidazole-1–hexanoic acid
(FFI), by using FFI–hexanoic acid (FFI-HA) (Fig 1
) as
the antigen, as well as antisera against AGEs in general, using
AGE-albumin as the antigen. The present article characterizes the
resulting antisera and provides immunocytochemical evidence for the
occurrence of FFI-like epitopes and other AGE epitopes in
atherosclerotic lesions of euglycemic WHHL rabbits. Furthermore, we
show that IgA, IgG, and IgM autoantibodies that recognize FFI-protein
adducts are present in the sera of human diabetic and control
subjects, thereby providing additional evidence for the formation of
FFI-like epitopes in vivo.
|
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Isolation of LDL
LDL (1.025 to 1.070 g/mL) was prepared from male guinea pigs by sequential ultracentrifugation, as previously described.43 The isolated lipoprotein was extensively dialyzed against phosphate-buffered saline (PBS; 0.15 mol/L NaCl, 36 mmol/L phosphate buffer, pH 7.35) containing 0.27 mmol/L EDTA. The protein content was determined by the method of Lowry et al,44 and LDL was sterile-filtered for further use.
Preparation of Immunogens and Competitors for Radioimmunoassays
(RIAs)
Conjugation of FFI-HA to proteins was performed with a
modification of the method described by Gendloff et al.45
FFI-HA was kindly provided by Dr Michael Yamin. FFI-HA was conjugated
to guinea pig LDL, guinea pig albumin, or keyhole limpet hemocyanin
(KLH) as follows. FFI-HA (1 mg, or 2.92 µmol) was added to equimolar
amounts of N,N'-dicyclohexylcarbodiimide (Pierce Chemical
Co) and N-hydroxysuccinimide (Pierce) in 0.1 mL
tetrahydrofuran (Pierce) and gently mixed for 24 hours at room
temperature. N-hydroxysuccinimide enhances the coupling
reaction between the hapten and the protein mediated by the
water-soluble carbodiimide. Precipitates were removed by filtration
through a 0.45-µm filter. The reaction mixture was washed with
tetrahydrofuran, dried under nitrogen, and resolubilized in 200 µL
dimethylformamide per milligram of FFI. Before use, LDL was extensively
dialyzed against 0.1 mol/L NaHCO3, pH 7.35,
containing 10 mg/mL EDTA (to avoid oxidative modification during the
procedure) and adjusted to 5 mg in 0.5 mL. The reaction mixture
containing FFI-HA was added dropwise to LDL under constant stirring (up
to a total of 200 µL or until excessive aggregation was observed) and
allowed to react for 24 hours in a light-proof vial. The conjugate was
then extensively dialyzed, first against 0.1 mol/L NaHCO3
with EDTA and then against PBS, pH 7.35, also containing 10 mg/mL EDTA.
The product of this reaction was termed FFI-LDL. Conjugation of FFI to
guinea pig albumin or KLH was performed similarly, also at a ratio of 1
mg FFI-HA per milligram of protein, and yielded FFI-albumin and
FFI-KLH, respectively.
AGE-modified proteins were also prepared according to the method of Stern and colleagues (Schmidt et al26 ). LDL, albumin, hemoglobin, and other proteins were incubated with either 500 mmol/L glucose, 500 mmol/L glucose-6-phosphate, or 250 mmol/L ribose in the presence of aprotinin (2 µg/mL), leupeptin (0.5 µg/mL), pepstatin (0.7 µg/mL), PMSF (1.5 mmol/L), EDTA (1 mmol/L), and BHT (1 mg/mL). Incubation of sterile-filtered solutions was carried out for 3 months at 37°C. Solutions were then extensively dialyzed against PBS and stored at 4°C. Malondialdehyde (MDA)-LDL and copper-oxidized LDL (Cu2+-LDL) were prepared as described.43
Immunization Procedures
Guinea pigs were immunized with FFI-HA conjugated to guinea pig
LDL or albumin. The rationale for the choice of these proteins was the
observation that homologous LDL is a powerful vehicle for
presenting an immunogen and results in the formation of antisera
specific for the modified lysine residues only; ie, the antibodies
recognize modified lysine on a number of different
proteins.46 Homologous protein was used to avoid the
generation of antibodies against epitopes of the native protein. In
addition, we also immunized guinea pigs with FFI-HA conjugated to KLH.
Antibodies against epitopes of KLH, the carrier protein, should not
recognize mammalian proteins. Finally, we immunized guinea pigs with
AGE-albumin. Two guinea pig antisera were generated for each antigen.
The primary immunization usually consisted of 150 µg protein/0.5 mL
PBS in Freund's complete adjuvant, and booster immunizations were
performed biweekly with 100 µg protein in Freund's incomplete
adjuvant, as previously described.43 Antibody titers were
determined in the preimmune sera and 5 to 7 days after the second and
subsequent boosts.
Determination of Antibody Titers and Specificity
Titers and specificity of both induced antibodies and
autoantibodies were determined by solid-phase RIA techniques, as
previously described.43 46 Antigen was coated at 5 µg/mL
for 2 hours at 37°C. The amount of bound immunoglobulin was
quantitated with a radiolabeled secondary antibody; eg, guinea pig IgG
was detected by goat anti–guinea pig IgG (OrganonTeknika–Cappel)
labeled at 
10 000 cpm/ng with 125I, using
lactoperoxidase (Enzymobeads, Bio-Rad Laboratories). The plates were
incubated for 4 hours at 4°C with 400 000 cpm per well of the
secondary antibody. Results were expressed as antibody binding as a
function of antibody dilution. Titers were defined as the reciprocal of
the highest dilution that gave binding values exceeding three times
that of the preimmune control. Competitive solid-phase RIAs were
performed similarly except that the antigen was plated at 1 or 2
µg/mL. A limiting and fixed dilution of the primary antibody (25
µL) was then added together with an equal volume of dilution buffer
(3% bovine serum albumin, 0.02% NaN3, 0.05% Tween
20, and 0.001% aprotinin in PBS) containing increasing amounts of
potential competitors. The results were calculated as
B/B0, ie, the amount of antibody bound to the plated
antigen in the presence of competitor (B) divided by the binding in the
absence of competitor (B0).
Competitive solid-phase RIAs were used to test the ability of antibodies that were generated against FFI-protein adducts to recognize epitopes on "physiologically" formed AGEs. AGEs were initially generated by incubation of LDL or albumin with 100 mmol/L glucose in the presence of EDTA under sterile conditions, in the dark, at 37°C for periods of 2 to 9 months. Aliquots of these preparations were also subjected to proteinase K digestion, acid hydrolysis, or acid hydrolysis and subsequent reaction with ammonia.47 Enzymatic digestion was carried out by adding proteinase K (Sigma Chemical Co) to LDL or albumin at a protein to enzyme ratio of 10:1 and incubating the resulting mixture at 37°C for 40 hours. Proteinase digestion was stopped by addition of 0.1 mol/L PMSF (Sigma) in ethanol to a final concentration of 1 mmol/L. Acid hydrolysis was performed by incubating the proteins with 6N HCl for 16 hours at 110°C. Samples were then dried under nitrogen, washed four times with distilled water, and resuspended in PBS.
Determination of Autoantibodies
The sera of 40 well-characterized diabetic patients (19 men and
21 women; age range, 19 to 82 years; mean age, 37 years; type of
diabetes, 12 type I and 28 type II; range of duration of diabetes, 0.2
to 35 years; mean duration, 12.2 years; HbA1c, 8.7±2.0%) and 40 age-
and sex-matched euglycemic control subjects (17 men and 23 women; age
range, 16 to 80 years; mean age, 39 years; HbA1c, 5.2±0.4%) were
tested for autoantibodies to FFI-LDL, plated as the antigen in
solid-phase RIAs. A fixed dilution of the sera was used as the primary
antibody (1:8 dilution for IgA and IgG and 1:256 dilution for IgM
autoantibodies), and the amount of autoantibody bound to the plated
antigen was determined by using radiolabeled monoclonal anti-human IgM,
IgG, or IgA (Zymed) as the second antibody. Results of these assays
were expressed as counts of second antibody bound to the human
autoantibodies. For each serum sample, a control value was subtracted,
which was determined in a duplicate set of wells without a specific
antigen. Selected sera from both control and diabetic subjects were
also subjected to competitive RIA to determine the specificity of
antibody binding to FFI-LDL. Student's t test was used to
compare autoantibody concentrations between diabetic and control
subjects, and ANOVA was used to test for correlations between antibody
concentrations and clinical parameters.
Tissue Preparation and Immunocytochemistry
Aortas of WHHL rabbits were perfusion-fixed at physiological
pressure with formal sucrose (4% paraformaldehyde, 5% sucrose, 20
mmol/L BHT, and 1 µmol/L EDTA, pH 7.4) as described.48
After dissection, the aortas were subjected to immersion-fixation for
an additional 12 hours. Aortas of control and 28-week alloxan diabetic
Lewis rats were flash-frozen in liquid nitrogen; portions of these
samples were subsequently fixed in paraformaldehyde and embedded in
paraffin.
Serial 8-µm-thick sections on microscope slides were rehydrated and
immunostained with an avidin/biotin/alkaline phosphatase system (Vector
Labs) as previously described.48 49 The primary antisera
against epitopes of AGEs that were used are listed in the
Table. In addition, some sections were stained with the
respective preimmune sera. Arterial sections of atherosclerotic WHHL
aortas were also stained with antisera and monoclonal antibodies
against epitopes generated during the oxidative modification of LDL,
ie, MDA2 (specific for MDA-lysine) and NA59 (specific for
4-hydroxynonenal–lysine).40 43 Controls included MB47, a
monoclonal antibody against native apo B-10050 ; RAM-11, a
macrophage-specific monoclonal antibody51 ; and nonspecific
sera. Competitive immunostaining was performed by preincubating the
antibody with excess potential competitor for 1 hour before
immunostaining and by comparing the resultant staining to that obtained
with the same final dilution of antibody in the absence of competitor.
To rule out nonspecific binding to the competitor, controls included
comparative immunostaining with a nonspecific antibody preincubated
with the same competitor (eg, staining with RAM-11, a
macrophage-specific monoclonal antibody, should not be affected by
preincubation with AGE-LDL).
|
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Characterization of Antisera to FFI-Protein Adducts
Even minor modification of lysine residues renders LDL highly immunogenic,46 and previous studies from this laboratory have shown that immunization of guinea pigs with modified homologous LDL and albumin resulted in monospecific antisera that recognized the adduct formed but not the native protein.52 In the present experiments, we wanted to generate antisera that would recognize FFI conjugated to proteins via lysines, as such FFI-lysine conjugates have been hypothesized to represent one of the structures generated during AGE formation. We used FFI-HA (Fig 1
) as a
model of FFI conjugated to lysine53 and covalently linked
this to lysine residues of LDL by using a modification of the
carbodiimide method described by Gendloff et al,45 which
directly links the carboxy-terminal carbon of HA to the 
-amino group
of lysine. Thus, this coupling reaction does not generate any
intermediary "linker" between the hapten and carrier, as occurs
when haptens are linked via the commonly used glutaraldehyde method.
(In fact, in preliminary experiments with glutaraldehyde as the linker,
we generated antisera that contained a population of antibodies that
recognized glutaraldehyde-modified LDL.) It should be kept in mind,
though, that FFI-HA itself models FFI-lysine. Immunizations of guinea
pigs with FFI-HA conjugated with homologous LDL (n=2), homologous
albumin (n=2), and KLH (n=2) yielded high titers of antibodies that
bound to both FFI-LDL and the respective antigen (Table). These
antisera showed no significant antibody titer against native,
unmodified LDL or albumin. To determine the specificity of each antiserum, we performed classic competition RIAs. In these assays, the various immunogens (ie, FFI-LDL, FFI-albumin, or FFI-KLH) were plated as the solid-phase reactant in wells of microtiter plates, and fixed and limiting dilutions of antisera were added in the absence or presence of increasing concentrations of FFI-HA or FFI-modified proteins. In these assays, equal amounts (by mass) of the modified proteins were added because we have no accurate method to measure their absolute FFI-lysine content. Thus, the assays give evidence of the specificity (ability to compete) and of the relative affinity (degree of parallelism of the slopes of the competition curves) of the antisera but do not allow quantitative comparisons between competitors, as the modified proteins contain different absolute numbers of FFI-lysine adducts.
As shown in Fig 2, FFI-HA was able to completely compete
for the binding of each antiserum to its respective immunogen. In other
words, FFI-HA completely prevented the binding of antiserum FL-1 to
FFI-LDL or of FA-1 to FFI-albumin. Native LDL, when subjected to a
"mock" conjugation (ie, when exposed to the same conditions used
to prepare FFI-LDL but in the absence of FFI-HA), did not compete (data
not shown). Furthermore, neither OxLDL nor MDA-LDL competed for binding
to antiserum FL-1. Together, these data clearly demonstrate that the
antisera recognize FFI-HA alone as well as FFI-HA conjugated to the
carrier protein used for immunization.
|
To document that each antiserum would also recognize FFI-HA when
conjugated to other proteins, additional competitive assays were
performed. Fig 3 shows recognition of various
glycosylated and nonglycosylated proteins by guinea pig antiserum FL-1,
which was generated by immunization with homologous FFI-LDL. Clearly,
this antiserum recognized FFI-protein adducts, irrespective of the
protein to which the FFI-HA was bound, ie, FFI-LDL, FFI-KLH, or
FFI-albumin (Fig 3A). Although exact quantitation cannot be extracted
from these data (as noted above), it nevertheless appears that the FFI
present on albumin was "seen" better by the antisera than was
the FFI present on LDL. This could be due to the fact that many of
the modified lysine residues on FFI-LDL were located at sites
inaccessible to the antibody or that were sterically hindered, ie, by
lipid. In contrast, modified lysine sites on albumin, a much smaller
and simpler protein, are more likely to be readily available in
solution to the antibodies. The fact that the competition curves are,
in general, parallel implies that those FFI-lysine residues available
to the antibodies are recognized with approximately equal affinity.
|
Although FFI was identified as an AGE,53 54 the natural
occurrence of FFI has been questioned, as it may be formed during
extraction and purification procedures as a by-product of acid
hydrolysis of glycated proteins in the presence of
ammonia.47 55 56 57 We therefore tested whether the
antibodies generated against FFI-LDL would also recognize more
generalized AGE preparations generated by long-term incubations of LDL
or serum albumin with glucose. The ability of the FFI-specific antisera
to recognize these AGE proteins showed considerable variability:
AGE–human serum albumin (AGE-HSA) was not usually recognized (Fig 3A).
Some AGE-LDL preparations were recognized to a modest extent, albeit
only when added in very high concentrations (Fig 3B). However, when
AGE-LDL or AGE-HSA was subjected to proteinase digestion or acid
hydrolysis (in the absence of ammonia), there was increased recognition
of the resulting breakdown products. Presumably, the latter
treatments expose epitopes otherwise sterically hindered from
interacting with the antibodies. The reaction with ammonia appeared to
increase immune recognition of some AGE-HSA preparations reported as
previously by Horiuchi et al47 but did not enhance immune
recognition of the initial FFI-LDL preparations (Fig 3B). By
contrast, several AGE proteins prepared with extremely
high concentrations of glucose, glucose-6-phosphate, or ribose
(250 to 500 mmol/L; see "Methods") contained epitopes recognized
by FL-1 (Fig 4A). This demonstrates that with very high
concentrations of sugars, sufficient FFI-lysine adducts were
formed to enable detection by FL-1, FA-1, and FK-1, even
without acid hydrolysis or protein digestion.
|
Competition assays with FL-1 also demonstrated that the epitopes
formed during oxidative modification of LDL were not
recognized by FL-1. As shown in Fig 4B, MDA-LDL and
Cu2+-LDL competed only minimally when added at high
concentration, and competition observed with these forms of OxLDL was
not significantly greater than that seen with native LDL.
The specificities of the antisera generated with FFI-albumin (FA-1) or
with FFI-KLH (FK-1) were similar to that of FL-1. These antisera
recognized not only FFI-HA alone (Fig 2) and the respective immunogens
but also other FFI-protein adducts (Fig 5A and 5B).
|
Characterization of Antisera to AGE-Albumin
Immunization with AGE-albumin induced the generation of high-titer
antisera (GPA-1 and GPA-2), which recognized AGE-albumin. However,
FFI-LDL did not compete with the antisera for binding to AGE-albumin
(Fig 6). Thus, neither antiserum cross-reacted with the
FFI-lysine epitope.
|
Immunocytochemistry
To further address the question of the natural occurrence of FFI
or an FFI-like epitope, we used the three antisera generated against
FFI-LDL, FFI-albumin, and FFI-KLH for immunocytochemical analysis
of aortic atherosclerotic lesions of euglycemic WHHL rabbits (mean±SD
nonfasting blood glucose, 100±9.4 mg/dL, n=14). Fig 7
shows serial sections of a transitional atherosclerotic lesion stained
with FL-1 (A), FA-1 (B), and FK-1 (C). All three antisera recognized
FFI-lysine epitopes in almost identical distribution. By contrast,
nonlesioned aortic segments showed only very light and diffuse medial
staining (data not shown). Significant adventitial staining was found
in all sections studied.
|
Competition studies confirmed the specificity of staining for
FFI-lysine adducts: Fig 7D shows a serial section stained with FL-1
that had been preincubated with excess FFI-albumin. Specific staining
within the atherosclerotic lesion was virtually abolished.
Preincubation of FL-1 with native LDL or MDA-LDL did not affect
staining (data not shown). Competitive immunocytochemistry was also
used to demonstrate that the epitope recognized by FL-1 is indeed an
AGE epitope. Preincubation of FL-1 with AGE-LDL prepared by prolonged
incubation of LDL with a high concentration of glucose markedly reduced
the staining intensity (Fig 8B) compared with the
no-competitor control (Fig 8A). Similar results were also obtained with
AGE-LDL prepared with ribose and with AGE-albumin. By contrast,
preincubation of FL-1 with fresh LDL and glucose or with MDA-LDL showed
no significant competition. Control staining with RAM-11, a monoclonal
antibody against macrophages, was not affected by preincubation with
AGE-LDL (data not shown).
|
GPA-1, an antiserum raised against AGE-albumin, also recognized
specific epitopes in atherosclerotic lesions (Fig 8D) and yielded
staining patterns very similar to those of FFI-lysine–specific
antisera. To exclude the possibility that some GPA-1 staining might
result from low-titer autoantibodies to MDA-lysine in the sera of many
species17 or from nonspecific binding, serial sections
were stained with an identical dilution of preimmune serum from the
same animal. The section stained with preimmune serum was devoid of
specific staining (Fig 8C).
The staining pattern obtained with the different antisera to AGE
epitopes was remarkably similar to that obtained with antibodies
against epitopes generated during the oxidative modification of
lipoproteins (Fig 9). Panels A and D show an advanced
lesion from a WHHL rabbit aorta stained with two monoclonal antibodies
against "oxidation-specific" epitopes: NA59 (specific for
4-hydroxynonenal–lysine) and MDA2 (specific for MDA-lysine),
respectively. Panels B and C show sections that were stained with
antisera against AGE epitopes (FL-1 and GPA-1, respectively). At higher
magnification (Fig 10A and 10B), the FFI-specific
antibodies displayed a more extracellular and diffuse distribution
pattern and some staining of the media, as opposed to the more intense
macrophage-associated staining often observed in early atherosclerotic
lesions with oxidation-specific antibodies. Nevertheless, some intimal
macrophages showed intense staining with FL-1 (Fig 10A).
|
|
To determine whether qualitative differences in AGE staining
could be detected by our antibodies in tissues from a diabetic animal,
we also immunostained aortas from diabetic and control rats. Fig 11 shows the thoracic aorta from a control (A) and a
diabetic (B) rat stained with a 1:250 dilution of the antibody against
FFI-LDL. None of these sections displayed any intimal thickening or
other atherosclerotic lesions. Although the immunostaining
procedure provides only semiquantitative results, the more extensive
and intense staining of the diabetic specimen is apparent, particularly
in the entire media. These results were representative of
multiple sections examined from the aortas of two diabetic and four
euglycemic rats.
|
Autoantibodies to FFI in the Sera of Human Subjects
Nakamura et al38 demonstrated the presence of AGE
epitopes in cardiac tissue of diabetic subjects. Using our antisera
against FFI-like epitopes, we demonstrated the presence of these
epitopes in aortic and renal tissues of both euglycemic and diabetic
human subjects (data not shown). Because homologous modified LDL and
albumin were immunogenic in guinea pigs and because FFI-like epitopes
were observed in human tissues, we determined whether autoantibodies
that recognize FFI epitopes were present in the sera of 40
well-characterized diabetic patients and 40 matched euglycemic control
subjects. The majority of these serum samples had autoantibody titers
(Fig 12). No differences were found between diabetic
and control sera in the titers of IgG and IgM autoantibodies, but the
diabetic sera had significantly more IgA autoantibodies than did
controls (P<.0001). Competitive RIAs were performed with
selected sera from both diabetic and control subjects to show the
specificity of autoantibody binding to FFI-LDL. FFI-LDL, added as a
fluid-phase competitor, was able to compete for autoantibody binding to
an extent of 50% to 100% (data not shown). Native LDL or
mock-modified LDL (see above) did not compete. In this limited study,
IgA autoantibody concentrations of diabetic subjects were not
correlated with the type of diabetes, duration of diabetes, or
treatment. These data provide additional evidence that FFI-like
epitopes exist in vivo in human subjects.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Several studies reported the presence of AGE-modified proteins in the circulation.12 58 59 Increased AGE concentrations were described in tissues from diabetic subjects,59 60 61 62 and Nakamura and colleagues38 recently used an AGE-specific antiserum to show that AGEs occur in coronary atheromas of some human diabetic subjects. Because lipid peroxidation is thought to be an important component of the atherogenic process and because it could enhance AGE formation, we sought to determine whether AGE products could be detected in atherosclerotic lesions of euglycemic WHHL rabbits.
The structure of individual AGE products and their occurrence in vivo remain largely unknown. Proposed AGE components isolated from AGE proteins generated in vivo include FFI,53 54 pentosidine,61 and pyrroles.62 To document the presence of AGE structures, we generated several monospecific antisera against one model AGE compound, FFI, as well as antisera against AGE-albumin. Using all of these antisera for immunocytochemistry, we demonstrated the presence of AGE products and specifically that of FFI-like structures in atherosclerotic lesions of euglycemic rabbits.
There is controversy in the literature regarding the in vivo existence
of FFI as part of the AGE products. Some investigators reported
evidence in support of its presence,53 54 whereas other
data have suggested that FFI is formed ex vivo as a result of acid
hydrolysis in the presence of ammonia.55 56 57 Our antisera
recognized FFI-lysine or FFI-like epitopes on a number of untreated AGE
preparations obtained by incubation of proteins with high concentration
of sugars (Fig 4A). Other AGE proteins prepared with a lower
concentration of glucose were recognized only after hydrolysis or
proteinase K digestion (Fig 3). This could mean that some FFI-like
epitopes are present in intact proteins but are masked and are
exposed only after protein hydrolysis or enzyme
digestion.12 58 59 We also found that ammonia treatment of
acid hydrolysates of proteins increased immunorecognition by FFI
antisera, as suggested by Horiuchi et al.47 Moreover,
epitopes could also be exposed by proteinase K digestion, which should
not alter chemical structure in the same way. Makita et
al58 59 also found that similar digestion was needed to
maximize epitope exposure. Furthermore, the demonstration of FFI-like
epitopes in some untreated AGE proteins and the immunocytochemical
demonstration of FFI or FFI-lysine epitopes in atherosclerotic tissues
or diabetic aortas not subjected to any interventions clearly indicate
the presence in vivo of FFI or FFI-like structures. Immunoreactivity
observed in arterial tissues from WHHL rabbits showed a particular
abundance of these presumed AGE epitopes within atherosclerotic
lesions. Competition studies with FFI and FFI-modified proteins
demonstrated the specificity of the immunocytochemical identification
of FFI-like epitopes. The ability of AGE-modified proteins to inhibit
binding of FFI antisera to tissue sections (Figs 7 and 8) showed that
FFI itself or an immunologically closely related epitope is formed
during long-term incubation of proteins with various reducing sugars;
ie, FFI is a component of AGE.
GPA-1, an antiserum generated against AGE-albumin that did not
cross-react with FFI-modified proteins, also recognized epitopes in
atherosclerotic lesions (Figs 8D and 9C) and showed staining patterns
similar to those obtained with FFI-specific antisera. Together, these
data demonstrate that AGE-specific epitopes are formed in
atherosclerotic lesions of euglycemic rabbits and that FFI (or
structures that are immunochemically very closely related to FFI) is
one of these compounds.
When the aortas of 28-week diabetic rats were compared with controls, a
more intense staining with the FFI-lysine–specific antibodies was
observed in some diabetic aortas (Fig 11). Although immunostaining is
only semiquantitative, these findings would agree with the
increase in AGE as determined by biochemical methods in experimental
diabetes reported by other authors. For example, Monnier and
colleagues63 reported a twofold increase in
collagen-linked chromophores in galactose-fed rats. A similar increase
in pentosidine, another AGE component, was seen in the collagen of
diabetic patients with renal disease.64 Using a
radioreceptor assay, Makita and colleagues59 also reported
that increases in AGE-modified proteins were detected in arterial
collagen of diabetic patients, and Nakamura et al38 found
AGE in the arteries of diabetic human subjects but not in those of
nondiabetic individuals.
When atherosclerotic lesions of WHHL rabbits were studied, we observed
a striking coincidence of staining with antibodies directed at
glycosylation end products and antibodies directed at
oxidation-specific epitopes. The general distribution of immunostaining
obtained with antibodies to epitopes typically generated during
oxidative modification of LDL, eg, MDA-lysine and
4-hydroxynonenal–lysine, was very similar to that obtained with
AGE-specific antibodies (Fig 9). Immunocytochemistry with FFI-specific
antibodies showed more extracellular, diffuse, and reticular staining
in earlier lesions than with oxidation-specific antibodies and some
staining of the media. Nevertheless, the similarity of the intense
staining of the "shoulder" areas of lesions (rich in both
macrophage/foam cells and lipids) and of the adventitia is of great
interest. Cross-reactivity between the antisera to FFI and AGE and
oxidation-specific antisera is most unlikely, given the very different
chemical nature of the antigens used to prepare the antisera. The
results of our competitive RIAs support this idea (Fig 4B).
Furthermore, by including FFI-albumin and FFI-KLH among the antigens
used to generate antisera against FFI-like structures, we minimized the
probability of generating antibodies against lipid peroxidation
epitopes. The colocalization of oxidation-specific adducts and
AGE-specific epitopes within the intimal lesion could be due in part to
cross-linking of lipoproteins by AGE,15 which would
prolong the half-life of LDL in the artery wall and expose it to
further oxidative modification by arterial macrophages or smooth muscle
cells. It is also possible that in vivo, certain immunologically
related epitopes may be formed during oxidation of LDL and the
oxidative reactions involved in the formation of AGE.29 30
Perhaps more likely is the possibility that products of lipid
peroxidation may enhance AGE formation, which may in turn enhance lipid
peroxidation (reviewed in References 65 and 6665 66 ). For example, products
of lipid peroxidation have been reported to increase the cross-linking
of nonenzymatically glycosylated collagen in vitro, and conversely,
glucose and glycosylated collagen have been reported to increase
free-radical production and to catalyze lipid
peroxidation.12 31 32 33 34 In addition, low-molecular-weight
peptides resulting from breakdown of AGE proteins are found in the
circulation and bind to LDL,67 and conceivably these could
also enhance the susceptibility of LDL to oxidation. Thus, lipid
peroxidation and formation of Amadori products and AGEs may be
intricately linked and could explain the apparent colocalization of the
immunostaining patterns in lipid-rich atherosclerotic lesions, even in
nondiabetic animals.
Although colocalization is consistent with the hypothesized mutual enhancement of oxidation and AGE formation, the increased AGE formation in atherosclerotic lesions of LDL receptor–deficient rabbits does not provide conclusive evidence, as both lipoprotein oxidation and AGE formation may independently result from increased generation of free radicals in this animal model. For example, hypercholesterolemia per se may increase endothelial generation of superoxide anion.68
Even though the simple covalent attachment of a sugar to autologous protein would not be expected to be immunogenic, autoantibodies that are prevalent in the sera of normal and diabetic human subjects recognize both Amadori products69 and epitopes of AGE.11 Autoantibodies that recognize several forms of OxLDL, in particular MDA-lysine, are prevalent in humans and other species.40 49 These autoantibodies are capable of binding to epitopes of OxLDL in lesions,48 and immunoglobulins isolated from atherosclerotic lesions, recognize OxLDL, which may be present in lesions as immune complexes with OxLDL.70 Such autoantibodies may not only be markers of protein modification but also modulators of atherogenesis by forming immune complexes with their respective antigens in the circulation or within the arterial wall. In addition, cell-mediated immunity may also occur. Increasing evidence indeed suggests an important role for the immune system in the atherogenic process.71 72
In the limited study of diabetic and control sera, no differences in titers of autoantibodies against FFI-LDL of IgG and IgM were found, but diabetic subjects showed significantly higher concentrations of autoantibodies of IgA. Because titers of circulating autoantibodies reflect genetic control of the response to a given antigen as well as variations in the production and consumption of antibodies, the interpretation of antibody titers is complex. The possible clinical relevance of this observation remains unclear and requires further testing in more rigidly matched patient populations. However, the fact that circulating autoantibodies to FFI-like structures were present in the sera of both normal and diabetic subjects provides further evidence for the in vivo occurrence of FFI or immunologically similar epitopes.
|
Acknowledgments |
|---|
This work was supported in part by National Heart, Lung, and Blood Institute grant HL14197 (Specialized Center of Research in Arteriosclerosis) and a gift from Alteon Inc. Dr Witztum was an Established Investigator of the American Heart Association during a part of this work. We thank Dr Sampath Parthasarathy for advice, Dr Michael Brownlee of the Albert Einstein College of Medicine, New York, NY, for generously supplying some of the diabetic rat tissues, and Mercedes Silvestre for excellent technical assistance.
|
Footnotes |
|---|
Reprint requests to Wulf Palinski, MD, Department of Medicine, 0682, University of California, San Diego, La Jolla, CA 92093.
Received October 20, 1994; accepted February 8, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Pyörälä K, Laakso M, Uusitupa M. Diabetes and arteriosclerosis: an epidemiologic view. Diabetes Metab Rev. 1987;3:463-524. [Medline] [Order article via Infotrieve]
2. Brownlee M. Glycation and diabetic complications. Diabetes. 1994;43:836-841. [Medline] [Order article via Infotrieve]
3. Cerami A, Vlassara H, Brownlee M. Role of advanced glycosylation products in complications of diabetes. Diabetes Care. 1988;11:73-79.
4. Njoroge FG, Monnier VM. The chemistry of the Maillard reaction under physiological conditions—a review. Prog Clin Biol Res. 1989;304:85-107. [Medline] [Order article via Infotrieve]
5. Monnier VM. Nonenzymatic glycosylation, the Maillard reaction and the aging process. J Gerontol (Biol Sci). 1990;45:B105-B111.
6. Baynes JW, Watkins NG, Fisher CI, Hull CJ, Patrick JS, Ahmed MU, Dunn JA, Thorpe SR. The Amadori product on protein: structure and reactions. Prog Clin Biol Res. 1989;304:43-67. [Medline] [Order article via Infotrieve]
7. Kato H, Hayase F, Dong BS, Oimomi M, Baba S. 3-Deoxyglucosone, an intermediate product of the Maillard reaction. Prog Clin Biol Res. 1989;304:69-84. [Medline] [Order article via Infotrieve]
8. Witztum JL, Mahoney EM, Branks MJ, Fisher M, Elam R, Steinberg D. Nonenzymatic glucosylation of low-density lipoprotein alters its biological activity. Diabetes. 1982;31:283-291. [Abstract]
9. Curtiss LK, Witztum JL. Plasma apolipoproteins A-I, A-II, B, C-I and E are glucosylated in hyperglycemic diabetes. Diabetes. 1985;34:452-461. [Abstract]
10. Steinbrecher UP, Witztum JL. Glucosylation of low density lipoproteins to an extent comparable to that seen in diabetes slows their catabolism. Diabetes. 1984;33:130-134. [Abstract]
11. Witztum JL, Koschinsky T. Metabolic and immunological consequences of glycation of low density lipoproteins. In: Baynes JW, Monnier VM, eds. The Maillard Reaction in Aging, Diabetes and Nutrition. New York, NY: Alan R Liss Inc; 1989:219-234.
12.
Bucala R, Makita Z, Koschinsky T, Cerami A, Vlassara H.
Lipid advanced glycosylation: pathway for lipid oxidation in
vivo. Proc Natl Acad Sci U S A. 1993;90:6434-6438.
13. Bucala R, Tracey KJ, Cerami A. Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilatation in experimental diabetes. J Clin Invest. 1991;87:432-438.
14. Brownlee M, Vlassara H, Cerami A. Nonenzymatic glycosylation products of collagen covalently trap low-density lipoprotein. Diabetes. 1985;34:938-941. [Abstract]
15.
Brownlee M, Pongor S, Cerami A. Covalent
attachment of soluble proteins by nonenzymatically glycosylated
collagen: role of the in situ formation of immune complexes.
J Exp Med. 1983;158:1739-1744.
16. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915-924. [Medline] [Order article via Infotrieve]
17. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991;88:1785-1792.
18. Palinski W. Lipoprotein oxidation: mechanisms and implications for atherogenesis. In: Born GVR, Schwartz CD, eds. New Horizons in Coronary Heart Disease. London, England: Current Science; 1993:13.1-13.11.
19.
Goldstein JL, Ho YK, Basu SK, Brown MS. Binding
site on macrophages that mediates uptake and degradation of acetylated
low density lipoprotein, producing massive cholesterol
deposition. Proc Natl Acad Sci U S A. 1979;76:333-337.
20.
Vlassara H, Brownlee M, Cerami A. High-affinity
receptor-mediated uptake and degradation of glucose-modified proteins:
a potential mechanism for the removal of senescent
macromolecules. Proc Natl Acad Sci U S A. 1985;82:5588-5592.
21.
Takata K, Horiuchi S, Araki N, Shiga M, Saitoh M,
Morino Y. Endocytic uptake of nonenzymatically glycosylated
proteins is mediated by a scavenger receptor for aldehyde-modified
proteins. J Biol Chem. 1988;263:14819-14825.
22. Radoff S, Cerami A, Vlassara H. Isolation of surface binding protein specific for advanced glycosylation end products from mouse macrophage-derived cell line RAW 264.7. Diabetes. 1990;39:1510-1518. [Abstract]
23. Neeper M, Schmid AM, Brett J, Yan SD, Wang F, Pan YC, Elliston K, Stern D, Shaw A. Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins. J Biol Chem. 1992;267:14988-15004.
24.
Schmidt AM, Mora R, Cao R, Yan SD, Brett J,
Ramakrishnan R, Tsang TC, Simionescu M, Stern D. The endothelial
cell binding site for advanced glycation end products consists of a
complex: an integral membrane protein and a lactoferrin-like
polypeptide. J Biol Chem. 1994;269:9882-9888.
25.
Kirstein M, Brett J, Radoff S, Ogawa S, Stern D,
Vlassara H. Advanced protein glycosylation induces
transendothelial human monocyte chemotaxis and secretion of
platelet-derived growth factor: role in vascular disease of diabetes
and aging. Proc Natl Acad Sci U S A. 1990;87:9010-9014.
26. Schmidt AM, Yan SD, Brett J, Mora R, Nowygrod R, Stern D. Regulation of human mononuclear phagocyte migration by cell surface-binding proteins for advanced glycation end products. J Clin Invest. 1993;91:2155-2168.
27.
Quinn MT, Parthasarathy S, Fong LG, Steinberg D.
Oxidatively modified low density lipoproteins: a potential role
in recruitment and retention of monocyte/macrophages during
atherogenesis. Proc Natl Acad Sci U S A. 1987;84:2995-2998.
28.
Yan SD, Schmid AM, Anderson GM, Zhang J, Brett J, Zou
YS, Pinsky D, Stern D. Enhanced cellular oxidant stress by the
interaction of advanced glycation end products with their
receptors/binding proteins. J Biol Chem. 1994;269:9889-9897.
29. Dunn JA, Patrick JS, Thorpe SR, Baynes JW. Oxidation of glycated proteins: age-dependent accumulation of N-epsilon-(carboxymethyl) lysine in lens protein. Biochemistry. 1989;28:9464-9468. [Medline] [Order article via Infotrieve]
30. Wolff SP, Bascal ZA, Hunt JV. "Autooxidative glycosylation": free radicals and glycation theory. In: Baynes JW, Monnier VM, eds. The Maillard Reaction in Aging, Diabetes and Nutrition. New York, NY: Alan R Liss Inc; 1989:259-278.
31. Mullarkey CJ, Edelstein D, Brownlee M. Free radical generation by early glycation products: a mechanism for accelerated atherogenesis in diabetes. Biochem Biophys Res Commun. 1990;173:932-939. [Medline] [Order article via Infotrieve]
32. Hicks M, Delbridge L, Yue, DK, Reeve TS. Catalysis of lipid peroxidation by glucose and glycosylated collagen. Biochem Biophys Res Commun. 1988;151:649-655. [Medline] [Order article via Infotrieve]
33. Hicks M, Delbridge L, Yue, DK, Reeve TS. Increase in crosslinking of nonenzymatically glycosylated collagen induced by products of lipid peroxidation. Arch Biochem Biophys. 1989;268:249-254. [Medline] [Order article via Infotrieve]
34. Hunt JV, Smith CCT, Wolff SP. Autoxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose. Diabetes. 1990;39:1420-1424. [Abstract]
35. Hunt JV, Bottoms MA, Clare K, Skamarauskas JT, Mitchinson MJ. Glucose oxidation and low-density lipoprotein-induced macrophage ceroid accumulation: possible implications for diabetic atherosclerosis. Biochem J. 1994;300:243-249.
36. Nishigaki I, Hagihara M, Tsunekawa H, Maseki M, Yagi K. Lipid peroxide levels of serum lipoprotein fractions of diabetic patients. Biochem Med. 1981;25:373-378. [Medline] [Order article via Infotrieve]
37. Morel DW, Chisolm GM. Antioxidant treatment of diabetic rats inhibits lipoprotein oxidation and cytotoxicity. J Lipid Res. 1989;30:1827-1834. [Abstract]
38. Nakamura Y, Horii Y, Nishino T, Shiiki H, Sakaguchi Y, Kagoshima T, Dohi K, Makita Z, Vlassara H, Bucala R. Immunocytochemical localization of advanced glycosylation end products in coronary atheroma and cardiac tissue in diabetes mellitus. Am J Pathol. 1993;143:1649-1656. [Abstract]
39.
Haberland ME, Fong D, Cheng L.
Malondialdehyde-altered protein occurs in atheroma of Watanabe
heritable hyperlipidemic rabbits. Science. 1988;241:215-218.
40.
Palinski W, Rosenfeld ME, Ylä-Herttuala S,
Gurtner GC, Socher SA, Butler S, Parthasarathy S, Carew TE, Steinberg
D, Witztum JL. Low density lipoprotein undergoes oxidative
modification in vivo. Proc Natl Acad Sci U S A. 1989;86:1372-1376.
41. Boyd HC, Gown AM, Wolfbauer G, Chait A. Direct evidence for a protein recognized by a monoclonal antibody against oxidatively modified LDL in atherosclerotic lesions from a Watanabe Heritable Hyperlipidemic rabbit. Am J Pathol. 1989;135:815-826. [Abstract]
42. Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086-1095.
43.
Palinski W, Ylä-Herttuala S, Rosenfeld ME, Butler
S, Socher SA, Parthasarathy S, Curtiss LK, Witztum JL. Antisera
and monoclonal antibodies specific for epitopes generated during the
oxidative modification of low density lipoprotein.
Arteriosclerosis. 1990;10:325-335.
44. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:1831-1841.
45. Gendloff EH, Casale WL, Ram BP, Tai JH, Pestka JJ, Hart LP. Hapten-protein conjugates prepared by the mixed anhydride method: cross-reactive antibodies in heterologous antisera. J Immunol Methods. 1986;92:15-20.[Medline] [Order article via Infotrieve]
46. Steinbrecher UP, Fisher M, Witztum JL, Curtiss LK. Immunogenicity of homologous low density lipoprotein after methylation, ethylation, acetylation or carbamylation: generation of antibodies specific for derivatyzed lysine. J Lipid Res. 1984;25:1109-1116. [Abstract]
47.
Horiuchi S, Shiga M, Araki N, Takata K, Saitoh M,
Morino Y. Evidence against in vivo presence of
2-(2-furoyl)-4(5)-(2furanyl)-1H-imidazole, a major fluorescent advanced
end product generated by nonenzymatic glycosylation. J
Biol Chem. 1988;263:18821-18826.
48.
Rosenfeld ME, Palinski W, Ylä-Herttuala S, Butler
S, Witztum JL. Distribution of oxidation-specific lipid-protein
adducts and apolipoprotein B in atherosclerotic lesions of varying
severity from WHHL rabbits. Arteriosclerosis. 1990;10:336-349.
49.
Palinski W, Ord V, Plump AS, Breslow JL, Steinberg D,
Witztum JL. ApoE-deficient mice are a model of lipoprotein
oxidation in atherogenesis: demonstration of oxidation-specific
epitopes in lesions and high titers of autoantibodies to
malondialdehyde-lysine in serum. Arterioscler
Thromb. 1994;14:605-616.
50.
Young SG, Witztum JL, Casal DC, Curtiss LK, Bernstein
S. Conservation of the low density lipoprotein receptor-binding
domain of apoprotein B: demonstration by a new monoclonal antibody,
MB-47. Arteriosclerosis. 1986;6:178-188.
51. Tsukada T, Rosenfeld ME, Ross R, Gown AM. Immunocytochemical analysis of cellular components of lesions of atherosclerosis in Watanabe and fat-fed rabbits using monoclonal antibodies. Arteriosclerosis. 1986;6:601-613. [Abstract]
52.
Witztum JL, Steinbrecher UP, Fisher M, Kesaniemi A.
Nonenzymatic glucosylation of homologous LDL and albumin render
them immunogenic in the guinea pig. Proc Natl Acad Sci
U S A. 1983;80:2757-2761.
53.
Pongor S, Ulrich PC, Bencsath FA, Cerami A.
Aging of proteins: isolation and identification of a fluorescent
chromophore from the reaction of polypeptides with glucose.
Proc Natl Acad Sci U S A. 1984;81:2684-2688.
54.
Njoroge FG, Fernandes AA, Monnier VM. Mechanism
of formation of the putative advanced glycosylation end product and
protein cross-link 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole.
J Biol Chem. 1988;263:10646-10652.
55.
Chang JCF, Ulrich PC, Bucala R, Cerami A.
Detection of an advanced glycosylation product bound to protein
in situ. J Biol Chem. 1985;260:7970-7974.
56. Gerhardinger C, Lapolla A, Crepaldi G, Fedele D, Ghezzo E, Seraglia R, Traldi P. Evidence of acid hydrolysis as responsible for 2-(2 furoyl)-4(5)-(2 furanyl)-1H-imidazole (FFI) production. Clin Chim Acta. 1990;189:335-340. [Medline] [Order article via Infotrieve]
57. Lapolla A, Gerhardinger C, Ghezzo E, Seraglia R, Sturaro A, Crepaldi G, Fedele D, Traldi P. Identification of furoyl-containing advanced glycation products in collagen samples from diabetic and healthy rats. Biochim Biophys Acta. 1990;1033:13-18. [Medline] [Order article via Infotrieve]
58.
Makita Z, Vlassara H, Cerami A, Bucala R.
Immunocytochemical detection of advanced glycosylation end
products in vivo. J Biol Chem. 1992;267:5133-5138.
59. Makita Z, Radoff S, Rayfield EJ, Yang Z, Skolnik E, Delaney V, Friedman EA, Cerami A, Vlassara H. Advanced glycosylation end products in patients with diabetic nephropathy. N Engl J Med. 1991;325:836-842. [Abstract]
60.
Hammes HP, Martin S, Federlin K, Geisen K,
Brownlee M. Aminoguanidine treatment inhibits the development of
experimental diabetic retinopathy. Proc Natl Acad Sci
U S A. 1991;88:11555-11558.
61.
Sell DR, Monnier VM. Structure elucidation of a
senescence cross-link from human extracellular matrix. J
Biol Chem. 1989;264:21597-21602.
62. Hayase F, Nagaraj RH, Miyata S, Njoroge FG, Monnier VM. Aging of proteins: immunological detection of a glucose-derived pyrrole formed during Maillard reaction in vivo. J Biol Chem. 1989;263:3758-3764.
63. Monnier VM, Sell DR, Abdul-Karim FW, Emancipator SN. Collagen browning and cross-linking are increased in chronic experimental hyperglycemia. Diabetes. 1988;37:867-872. [Abstract]
64. Sell DR, Monnier VM. End-stage renal disease and diabetes catalyze the formation of a pentose-derived crosslink from aging human collagen. J Clin Invest. 1990;85:380-384.
65. Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes. 1991;40:405-412. [Abstract]
66. Lyons TJ. Glycation and oxidation: a role in the pathogenesis of atherosclerosis. Am J Cardiol. 1993;71:26B-31B. [Medline] [Order article via Infotrieve]
67.
Bucala R, Makita Z, Vega G, Grundy S, Koschinsky T,
Cerami A, Vlassara H. Modification of low density lipoprotein by
advanced glycation end products contributes to the dyslipidemia of
diabetes and renal insufficiency. Proc Natl Acad Sci
U S A. 1994;91:9441-9445.
68. Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest. 1993;91:2546-2551.
69.
Witztum JL, Steinbrecher UP, Kesaniemi YA, Fisher M.
Autoantibodies to glucosylated proteins in the plasma of
patients with diabetes mellitus. Proc Natl Acad Sci
U S A. 1984;81:3204-3208.
70.
Ylä-Herttuala S, Palinski W, Butler S, Picard S,
Steinberg D, Witztum JL. Rabbit and human atherosclerotic
lesions contain IgG that recognizes epitopes of oxidized low density
lipoprotein. Arterioscler Thromb. 1994;14:32-40.
71. Libby P, Hansson GK. Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991;64:5-15. [Medline] [Order article via Infotrieve]
72.
Palinski W, Miller E, Witztum JL. Immunization
of LDL receptor-deficient rabbits with homologous
malondialdehyde-modified LDL reduces atherogenesis. Proc
Natl Acad Sci U S A. 1995;92:821-825.
This article has been cited by other articles:
 |
|
 |
|
  M. Alikhani, C. M. MacLellan, M. Raptis, S. Vora, P. C. Trackman, and D. T. Graves Advanced glycation end products induce apoptosis in fibroblasts through activation of ROS, MAP kinases, and the FOXO1 transcription factor Am J Physiol Cell Physiol, February 1, 2007; 292(2): C850 - C856. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Alikhani, M. Alikhani, C. M. Boyd, K. Nagao, P. C. Trackman, and D. T. Graves Advanced Glycation End Products Enhance Expression of Pro-apoptotic Genes and Stimulate Fibroblast Apoptosis through Cytoplasmic and Mitochondrial Pathways J. Biol. Chem., April 1, 2005; 280(13): 12087 - 12095. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. S. Lim, R. J. MacFadyen, and G. Y. H. Lip Diabetes Mellitus, the Renin-Angiotensin-Aldosterone System, and the Heart Arch Intern Med, September 13, 2004; 164(16): 1737 - 1748. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Virella, S. R. Thorpe, N. L. Alderson, E. M. Stephan, D. Atchley, F. Wagner, and M. F. Lopes-Virella Autoimmune response to advanced glycosylation end-products of human LDL J. Lipid Res., March 1, 2003; 44(3): 487 - 493. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Brizzi, P. Dentelli, R. Gambino, S. Cabodi, M. Cassader, A. Castelli, P. Defilippi, L. Pegoraro, and G. Pagano STAT5 Activation Induced by Diabetic LDL Depends on LDL Glycation and Occurs Via src Kinase Activity Diabetes, November 1, 2002; 51(11): 3311 - 3317. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Miyata, C. van Ypersele de Strihou, Y. Ueda, K. Ichimori, R. Inagi, H. Onogi, N. Ishikawa, M. Nangaku, and K. Kurokawa Angiotensin II Receptor Antagonists and Angiotensin-Converting Enzyme Inhibitors Lower In Vitro the Formation of Advanced Glycation End Products: Biochemical Mechanisms J. Am. Soc. Nephrol., October 1, 2002; 13(10): 2478 - 2487. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Collison, R. S. Parhar, S. S. Saleh, B. F. Meyer, A. A. Kwaasi, M. M. Hammami, A. M. Schmidt, D. M. Stern, and F. A. Al-Mohanna RAGE-mediated neutrophil dysfunction is evoked by advanced glycation end products (AGEs) J. Leukoc. Biol., March 1, 2002; 71(3): 433 - 444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bierhaus, S. Schiekofer, M. Schwaninger, M. Andrassy, P. M. Humpert, J. Chen, M. Hong, T. Luther, T. Henle, I. Kloting, et al. Diabetes-Associated Sustained Activation of the Transcription Factor Nuclear Factor-{kappa}B Diabetes, December 1, 2001; 50(12): 2792 - 2808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Mullick, B. A. Walsh, K. M. Reiser, and J. C. Rutledge Chronic estradiol treatment attenuates stiffening, glycoxidation, and permeability in rat carotid arteries Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H2204 - H2210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-P. Wautier, O. Chappey, S. Corda, D. M. Stern, A. M. Schmidt, and J.-L. Wautier Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE Am J Physiol Endocrinol Metab, May 1, 2001; 280(5): E685 - E694. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sakata, N. Uesugi, S. Takebayashi, R. Nagai, T. Jono, S. Horiuchi, M. Takeya, H. Itabe, T. Takano, T. Myint, et al. Glycoxidation and lipid peroxidation of low-density lipoprotein can synergistically enhance atherogenesis Cardiovasc Res, February 1, 2001; 49(2): 466 - 475. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Tsimikas, W. Palinski, and J. L. Witztum Circulating Autoantibodies to Oxidized LDL Correlate With Arterial Accumulation and Depletion of Oxidized LDL in LDL Receptor-Deficient Mice Arterioscler. Thromb. Vasc. Biol., January 1, 2001; 21(1): 95 - 100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M Crauwels, A. G Herman, and H. Bult Local application of advanced glycation end products and intimal hyperplasia in the rabbit collared carotid artery Cardiovasc Res, July 1, 2000; 47(1): 173 - 182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kislinger, C. Fu, B. Huber, W. Qu, A. Taguchi, S. Du Yan, M. Hofmann, S. F. Yan, M. Pischetsrieder, D. Stern, et al. Nepsilon -(Carboxymethyl)Lysine Adducts of Proteins Are Ligands for Receptor for Advanced Glycation End Products That Activate Cell Signaling Pathways and Modulate Gene Expression J. Biol. Chem., October 29, 1999; 274(44): 31740 - 31749. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Merat, F. Casanada, M. Sutphin, W. Palinski, and P. D. Reaven Western-Type Diets Induce Insulin Resistance and Hyperinsulinemia in LDL Receptor-Deficient Mice But Do Not Increase Aortic Atherosclerosis Compared With Normoinsulinemic Mice in Which Similar Plasma Cholesterol Levels Are Achieved by a Fructose-Rich Diet Arterioscler. Thromb. Vasc. Biol., May 1, 1999; 19(5): 1223 - 1230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Schmidt, S. D. Yan, J.-L. Wautier, and D. Stern Activation of Receptor for Advanced Glycation End Products : A Mechanism for Chronic Vascular Dysfunction in Diabetic Vasculopathy and Atherosclerosis Circ. Res., March 19, 1999; 84(5): 489 - 497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Du Yan, Y. Shi, A. Zhu, J. Fu, H. Zhu, Y. Zhu, L. Gibson, E. Stern, K. Collison, F. Al-Mohanna, et al. Role of ERAB/L-3-Hydroxyacyl-coenzyme A Dehydrogenase Type II Activity in Abeta -induced Cytotoxicity J. Biol. Chem., January 22, 1999; 274(4): 2145 - 2156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bierhaus, M. A Hofmann, R. Ziegler, and P. P Nawroth AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept Cardiovasc Res, March 1, 1998; 37(3): 586 - 600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bierhaus, T. Illmer, M. Kasper, T. Luther, P. Quehenberger, H. Tritschler, P. Wahl, R. Ziegler, M. Muller, and P. P. Nawroth Advanced Glycation End Product (AGE)–Mediated Induction of Tissue Factor in Cultured Endothelial Cells Is Dependent on RAGE Circulation, October 7, 1997; 96(7): 2262 - 2271. [Abstract] [Full Text]
|
|
|
|
|
|
P. Reaven, S. Merat, F. Casanada, M. Sutphin, and W. Palinski Effect of Streptozotocin-Induced Hyperglycemia on Lipid Profiles, Formation of Advanced Glycation Endproducts in Lesions, and Extent of Atherosclerosis in LDL Receptor-Deficient Mice Arterioscler. Thromb. Vasc. Biol., October 1, 1997; 17(10): 2250 - 2256. [Abstract] [Full Text]
|
|
|
|
|
|
J. Fruebis, V. Gonzalez, M. Silvestre, and W. Palinski Effect of Probucol Treatment on Gene Expression of VCAM-1, MCP-1, and M-CSF in the Aortic Wall of LDL Receptor–Deficient Rabbits During Early Atherogenesis Arterioscler. Thromb. Vasc. Biol., July 1, 1997; 17(7): 1289 - 1302. [Abstract] [Full Text]
|
|
|
|
|
|
M.-X. Fu, J.ús R. Requena, A. J. Jenkins, T. J. Lyons, J. W. Baynes, and S. R. Thorpe The Advanced Glycation End Product, N[IMAGE]-(Carboxymethyl)lysine, Is a Product of both Lipid Peroxidation and Glycoxidation Reactions J. Biol. Chem., April 26, 1996; 271(17): 9982 - 9986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Basta, G. Lazzerini, M. Massaro, T. Simoncini, P. Tanganelli, C. Fu, T. Kislinger, D. M. Stern, A. M. Schmidt, and R. De Caterina Advanced Glycation End Products Activate Endothelium Through Signal-Transduction Receptor RAGE: A Mechanism for Amplification of Inflammatory Responses Circulation, February 19, 2002; 105(7): 816 - 822. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||